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This work is dedicated to the memory of Dr. Koppal<a V. Rao, an extraordinary 
scientist, dedicated teacher and very dear friend. I feel blessed to have known and 
worked with him. 


Much of the work in this dissertation was done with the guidance and expertise of 
the late Dr. K. V. Rao, who passed away on February 20, 1998. Professor Rao and I 
had known each other since 1981 and I am grateful to him for encouraging me to return 
for graduate studies and for his true friendship with me. I would also like to thank his 
wife and children for their much appreciated support, friendship and encouragement. 

I wish to thank Dr. John Perrin for assuming the position of chairman of my 
supervisory committee and for his kind encouragement and guidance. He has helped 
me in many ways and I am very grateful to him for his persistence in pushing me to 
complete this work. I would also like to thank Dr. Margaret James, Dr. Jonathan Eric 
Enholm, Dr. Kenneth Sloan, and Dr. Stephen Schulman for participating on my 
supervisory committee and for their thoughtful advice and expertise. 

I wish to thank my mother and father for their kind encouragement and love, and 
also my three sisters, brothers-in-law, niece and nephews. I would like to thank the 
Graduate School and many other University of Florida personnel for all of the kind 
assistance they have provided, especially Gladys Jan Kalman and Nancy Rosa for all of 
their helpful assistance. 










Background of Research at the University of Florida 6 

Methods 6 


Earlier Studies 11 

Studies after the Discovery of Taxol 14 

Semi-synthesis of Taxol 17 

Total Synthesis 18 

Other Synthetic Approaches 20 

General Structural Features of Taxanes 24 

Taxa-4(20);11-dienes 25 

4(20)-Epoxides 25 

Oxetanes 26 

Abeotaxanes 28 


Fractionation of the Needles of Taxus brevifolia 29 

Brevitaxane A (Brevifoliol) [3-1] 31 

Hydroxyl Functionalities 33 

4/20 Unsaturation 36 

Number and Nature of the Oxygen Substitution 37 

Experimental 44 

Extraction of the Needles of Taxus brevifolia 44 

Reverse Phase Column Chromatography: 45 

Brevifoliol [3-1] 46 

Brevifoliol-5-Monoacetate [3-2] 48 


Brevifoliol-5,13-Diacetate[3-4] 49 

Brevifoliol-13-Ketone[3-6] 50 

Dihydrobrevifoliol [3-7] 51 

Brevifoliol Epoxide [3-8] 51 

Ozonization of Brevifoliol: Brevifoliol-norketone [3-9] 52 

Brevifoliol-4,20-Diol[3-10] 53 

Saponification of Brevifoliol [3-11] 54 

DebenzoyI Brevifoliol-Pentaacetate [3-12] 54 

Periodate Oxidation of [3-11] to [3-13] 55 

Formation of Osazone [3-14] from [3-13] with 2,4-DNPH 55 

DebenzoyI Brevifoliol [3-15] 56 


1. Acid-Catalyzed Acetylation 58 

2. Oxidation 59 

3. Action of BFs on Brevifoliol [4-3] 61 

4. Reaction with Iodine/Silver Acetate [4-4] 65 

Experimental 69 

Brevifoliol Triacetate [3-5] 69 

Oxidation with Jones Reagent to [4-1] 69 

Action of Boron Trifluoride on Brevifoliol [4-3] 70 

Reaction with Iodine and Silver Acetate [4-4] 70 

Acetylation of [4-4] to [4-5] 72 

Reaction with N-Bromosuccinimide and Silver Acetate [4-6] 72 

Reaction of [4-4] with N-Bromosuccinimide [4-7] 73 


Brevifoliol [3-1] 79 

Taxanes I [5-1] and II [5-2] 79 

Taxanelll[2-1] 80 

Taxane IV [2-2] 80 

Taxol [5-3] 80 

Ozonolysis of [2-2] 80 

Experimental 81 

Extraction: 81 

Chromatography: 81 

Characterization of the Taxane Components of Taxusx Media Hicksii 82 

Brevifoliol [2-1] 82 

Taxanes I and II [5-1] and [5-2] 83 

Taxane III [2-1] 84 

Taxane IV [2-2] 84 

Taxol [5-3] 85 

Ozonolysis of Compound [2-2] 86 


Taxiflorine 89 

Experimental 93 

Extraction 93 

Characterization of the Taxane Constituents of Taxus floridana 95 

lO-Deacety! Baccatin III [2-7] 95 

Brevifoliol [3-1] 95 

Taxiflorine[6-1] 96 

Baccatin VI [6-2] 96 

Taxol [5-3] 98 

Acetylation of Taxiflorine to [6-3] 98 

Benzoylation of Taxiflorine to [6-4] 98 

Saponification and Acetylation of [6-1] to [6-5] 99 


General 100 

Experimental 102 

Flavonoids 103 

Quercetin Rutoside (Rutin) 103 

Quercetin 104 

Sciadopitysin 104 

p-Sitosterol-p-D-Glucoside 105 

p-Sitosterol-(3-D-Glucoside Tetra-acetate 105 

p~Sitosterol 106 

Phytoecdysteroids 107 

Ecdysterone & 2p, 3p, 22a-Triacetate 107 

Ponasterone A and 2p, 3p, 22a Triacetate 107 

Phenolic Compounds 108 

Usnic Acid 108 

Betuloside (4-(4'-Hydroxyphenyl)-2R-butanol Glucoside)) & Aglycone 109 





Table page 

3-1 : Proton NMR Spectra of Brevifoliol and Brevifoliol Acetates 33 

3-2 : Carbon NMR Spectra of Brevifoliol and Brevifoliol Acetates 34 

4-1 : NMR Spectra of Compound [4-3] from BF3 Reaction 63 

6-1 : Proton NMR Spectra of Compounds [6-3], [6-4] and [6-5] 91 



Figure E^ 

2-1 : Early Studies on the Constituents of some Taxus Species 13 

2-2 : Taxol and some Synthetic Targets 16 

2-3 : Nicolaou's Retrosynthetic Strategy 18 

2-4 : Nicolaou's Taxane Ring Synthesis 21 

2-5 : Nicolaou's Final Synthetic Intermediates 22 

2-6 : Starting Points of Other Synthetic Strategies 23 

3-1 : Proton NMR Spectrum of Brevifoliol 32 

3-2 : Brevifoliol and Reaction Products 35 

3-3 : Brevifoliol Hexaol Reaction Products 39 

4-1 : Oxidation Products 61 

4-2 : BFs-etherate Catalyzed Elimination Product 64 

4-3 : DEPT Spectra of BF3 Elimination Product [4-3] 64 

4-4 : Iodine/Silver Acetate Product [4-4] and Acetate 66 

4-5 : H,H-COSY Spectrum of [4-4] 67 

4-6 : HETCOR Spectrum of [4-4] 68 

5-1 : Fractionation of the Extract of Taxus x media Hicksii Needles 77 

5-2 : HPLC Trace of Taxanes Coeluting with Taxol 78 

5-3 : Progress of Elution of Taxanes from Reverse Phase Column 78 

6-1 : Taxanes and Analogues from "Taxas x med/a Hicksii 92 


6-2 : Carbon NMR Spectrum of Baccatin VI 97 


Abstract of Dissertation Presented to the Graduate School 
of the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 




Richard M. Davies 

December 1998 

Chairman: Koppaka V. Rao 

Cochairman: John H. Perrin 

Major Department: Medicinal Chemistry 

Taxol is a promising antineoplastic agent originally reported in 1971 by Wani and 
Wall, isolated from the bark of the Pacific yew {Taxus brevifolia). Intensive research in 
the last decade has demonstrated that this drug possesses exceptional activity in the 
treatment of many difficult types of cancer. 

From the beginning taxol has proven to be a difficult compound to obtain, v\/ith 
very low yields and a highly complex structure with many chiral centers and sensitive 
moieties. Originally obtained from the bark of a very slow growing tree, the possibility of 
growing various Taxus (yew) species under hydroponic conditions has been investigated 

in this project. 

One local variety, known as Taxus floridana (Florida yew) was found to grow well 
and produce taxol and other useful taxanes. During initial investigations a simple and 
elegant method for the isolation of taxol using reverse phase bonded silica was 
developed. Generous funding by the University of Florida Division of Sponsored 

Research made possible the construction of a pilot plant scale facility where these 
isolation methods were successfully implemented. 

Excellent yields and the isolation of many related taxanes have proven that this 
method is superior to currently approved processes used in the production of taxol. The 
failure of other researchers to employ bonded silica gel for preparative columns in the 
past may reflect experiences with analytical columns, but this method has proven to be 
quite exceptional and should be employed extensively. 

This dissertation covers many crystalline and non-crystalline compounds isolated 
and characterized as a result of this project. Some results from the application of this 
technique for the isolation of taxanes from the needles of Taxus brevifolia, Taxus x 
media cultivar Hicksii, and T. floridana are presented. Similar experiments on the bark 
and wood of T. brevifolia are also described. 



During the late 1950s, the National Cancer Institute initiated a program with the 
objective of discovering compounds from natural sources, which might prove useful in 
the treatment of various human cancers. In this program, plant samples from various 
parts of the world were collected and brought to participating laboratories, where the 
active principles were isolated, chemically characterized, and subjected to testing in 
various murine tumors. 

It is in such a context that a sample of the barl< of the Pacific yew (Taxus 
brevifolia, Nutt.) was extracted and the active principle called taxol* was reported by 
Wani and coworkers in 1971 (Wani et al. 1971). Although it exhibited potent cytotoxicity 
in some tumor assays, many unsuccessful lead compounds also are cytotoxic in these 
assays. Taxol did not appear exceptional, and the problems of low yield and poor 
solubility discouraged the pursuit of further research for many years. During the late 
1970s, activity against B-16 melanoma in mice, and several human tumors grown in 
athymic mice was recognized (e.g. MX-1 human mammary xenograft). These activities 
rekindled interest in taxol as a candidate for cancer treatment, resulting in further studies 
and human clinical trials. In the ensuing years the pace of research into taxol and 
taxoids has increased dramatically. 

*Taxol® is a registered trademark since 1993 by Bristol Myers Squibb Co., with 
paclitaxel being the generic name. 'Taxol' will be used in this dissertation as the generic 
name, as this work was started before this change. 


A study of its mode of action revealed that it blocl<ed cell division at the cell cycle 
through its specific action on the G2/M phase of the tubulin/ microtubule system. Unlike 
other antitumor drugs such as colchicine, vincristine and vinblastine, which act as tubulin 
poisons, taxol exhibited a novel mode of action (Schiff et al. 1979). Microtubules are 
involved in the formation of the mitotic spindle fibers necessary for the replication of DNA 
and are also integral building blocks within the cell wall. They are generated from a 
protein known as tubulin, and a dynamic equilibrium exists between tubulin and 
microtubules in vivo. In the presence of taxol, the polymerization of tubulin produces 
what are now known as oligo-microtubules. In contrast to the usual microtubules, which 
can be readily disassembled, these oligo-microtubules resist disassembly to tubulin, 
thereby preventing cell division (Horwitz, 1992). 

Based on potent activity against important experimental tumors and its unique 
mode of action, interest in taxol became greatly enhanced, and it was approved for 
human Phase I clinical trials in the early 1980s. Taxol showed significant activity in 
human tumors in Phase I and Phase II clinical trials, especially in ovarian and breast 
carcinomas (McGuire e^ al. 1989; Holmes e^ al. 1991). The scientific community took a 
special interest in taxol at that time due to the lack of adequate treatment options 
available for ovarian cancer. 

This knowledge established taxol as an important antitumor drug and stimulated 
a renewed interest in it. Intensive worldwide studies have reached explosive proportions 
since 1994 concerning its production, chemistry, biochemistry and many other aspects. 

At that point, two problems needed solution before taxol could become a viable 
alternative as a useful treatment against any type of cancer. First, the lipophilic nature of 
taxol made it difficult to develop an acceptable dosage form for this drug. Gradually, this 
was overcome by the introduction of a suitable, "relatively" non-toxic dosage form. 


Interestingly, the poor solubility characteristics of taxol might prove to be 
responsible for new discoveries regarding a problem of cross resistance to different 
classes of chemotherapeutic agents, caused by non-specific drug efflux and referred to 
as the MDR phenotype. The MDR phenotype is a gene that has been linked to multiple 
drug resistance (hence MDR); and some studies indicate that the solvent used for the 
delivery of taxol might have good activity against this common cause for therapeutic 
failure in the treatment of cancer (Woodcock et al. 1990; Webster et al. 1993; Fjallskog 
et al. 1993). It is known that this effect can result from the expression of plasma- 
membrane transport proteins (P-glycoproteins) which can enhance the efflux of 
structurally unrelated compounds from the cancer cell. At least three reports suggest 
that the solvent Cremaphore LH might enhance the antitumor actions of taxol when the 
tumor(s) display the MDR phenotype, and further work with cremaphores alone and in 
combination with other antitumor agents is needed to clarify this seemingly serendipitous 


Cremaphore LH is a form of ethoxylated castor oil and is responsible for many 
adverse drug reactions during the administration of taxol, and pretreatment with 
corticosteroids and antihistamines if often required to prevent allergic response up to 
and including anaphylaxis and death. Perhaps more difficult than this solubility concern 
was the procurement of an adequate supply of taxol for clinical trials and the anticipated 
needs for subsequent worldwide clinical use. Reported yields of taxol from the dried 
bark of T. brevifolia were averaging around 0.01%. 

A large-scale process for the isolation of taxol was developed by Polysciences, 
Inc. (Paul Valley Industnal Park, Warrington, PA 18976); with yields of 0.005-0.01% 
(Boettner et al. 1979). Under these conditions, one kilogram of the bark could be 
expected to provide only 50-100 mg of taxol at best (or 30,000 lbs. being required for 
obtaining one Kg. of taxol). Approximately 2 grams of taxol are needed for one complete 

course of therapy for a patient and this translates into requiring the bark from five to ten 
trees based on such reported yields. 

Only a few studies on the taxol content of other species of Taxus were published 
from the time its importance was recognized in 1980 until 1992. From the bark of T. 
walllchiana were isolated taxol and the closely related cephalomannine, as well as other 
taxoids (Miller et al. 1981; Miller, 1980). From the bark of T. baccata L., Senilh et al. 
isolated nearly 20 different taxoids, including taxol, cephalomannine and a series of 
xyloside derivatives of these (Senilh et al. 1984), The taxoid content of the needles of 
Taxus baccata was studied and 10-deacetyl baccatin III isolated in relatively high yields 
(Chauviere et al. 1981). The fractions from the large scale (Polysciences) process from 
the bark of T. brevifolia were also investigated to recover any other taxoids with useful 
activity, or with possible semisynthetic utility such as conversion to taxol. However, only 
minute yields of 10-deacetylbaccatin III, 7-epitaxol and 10-deacetyl-10-oxotaxol were 
reported in these studies (Huang et al. 1986; Kingston et al. 1982); leaving a strong 
impression that the bark of T. brevifolia is a poor source for not only taxol, but also for 
any other useful analogues of taxol. 

In spite of these problems, the bark of T. brevifolia has been accepted as the 
primary source for taxol until recently. However, since at the expected demand for taxol 
and the yields that can be realized from the bark, the yew tree population would be 
depleted in a few years, the use of the bark must stop. Among the alternatives that were 
being, considered to avoid this prospect are the following: 1) the use of needles, which 
are a renewable source, 2) growing the plant in tissue culture, 3) semi-synthesis from 
approphate naturally occurring taxoids and 4) total synthesis. Progress has been made 
on all of these fronts. 

As far as the needles are concerned, the most important candidate selected for 
direct isolation of taxol is the ornamental yew {Taxus x media cultivar Hicksii). Other 

than analytical HPLC studies on the taxol content of the needles under various 
conditions, no practical methodology for the isolation of taxol or other taxoids has been 
published. Publications from this laboratory which address these issues are essentially 
the only work available in the literature (Rao et al. 1995; Rao et al. 1996). In addition to 
direct isolation of taxol, the needles were also examined for the presence of analogues 
such as 10-deacetyl baccatin III, since semi-synthesis from such is already an important 
alternative. The two most important species, T. baccata L. and T. wallichiana Zucc, 
have become the focus of attention since they were demonstrated to contain the highest 
concentrations of 10-deacetyl baccatin III. 

Growing of various tissues of T. brevlfolia in plant cell culture has been under 
development since 1990 and the methods have been standardized in many laboratories. 
However, the yields, as yet, have not been very attractive. Further research is expected 
to overcome this problem. Work on this alternative will continue due to the 
attractiveness of this approach and its potential for large-scale operations. 

Starting with 10-deacetyl baccatin III, considerable progress was made in the 
area of semi-synthesis. In the first recorded semi-synthesis of taxol, the 13-cinnamate 
ester of 7-protected baccatin III was converted to the phenyl isoserine ester through a 
Sharpless hydroxy-amination (Denis et al. 1988). At this point, as an alternative to 
benzoylation of the amino group that will yield taxol, a t-BOC group (tert- 
butoxycarbonyl) was introduced, along with leaving the 10-hydroxyl free, to obtain an 
analogue known as taxotere. On the basis of its activity, taxotere has also been 
approved as an antitumor drug. Two important schemes for preparing taxol from 10- 
deacetyl baccatin III have been well developed and used for the large scale semi- 
synthesis of taxol as discussed in Chapter 2 (Denis et al. 1994; Ojima et al. 1991; Holton 
etal. 1992). 

Two total syntheses of taxol have been recorded, and several other approaches 
tovi/ards the synthesis have also been reported in the literature as discussed briefly in 
Chapter 2 (Nicolaou et al. 1994a, 1994b, 1995a, 1995b, 1995c; Holton et al. 1994a, 
1994b). Although these methods demonstrate remarkable achievements in the field of 
synthetic organic chemistry, they do not offer a practical method for the large-scale 
production of taxol or its analogues at this time. 

Background of Research at the University of Florida 

As part of one of these alternative quests, the National Cancer Institute hoped 
that instead of using the bark of the Pacific yev^^, the plant should be grown under 
hydroponic conditions, and they wanted to know whether plants grown in this manner 
would produce enough taxol for isolation. This laboratory was approached with this idea 
in early 1990, and with collaboration from Prof. George Hochmuth, Jr., of I FAS, 
University of Florida, the project was started. More on this aspect will be discussed in 
Chapter 6. In order to learn the current knowledge concerning the analysis and isolation 
of taxol, the pertinent literature was consulted. This yielded only a few papers on the 
isolation of taxol and taxoids, which were outlined above. 


In general, the methods were found to be too cumbersome for others to repeat. 
For example, one of these publications in which isolation of taxol and its analogues was 
described from T. wallichiana, used the following steps, starting with the concentrated 
ethanolic extract of the plant: 

1 . Partition between water and hexane 

2. Extraction of the aqueous phase with chloroform 

3. Silica gel chromatography on the chloroform extract 

4. A second silica gel chromatography 

5. Counter-current distribution 

6. HPLC on the appropriate fractions 

7. A second HPLC on the appropriate fractions 

Similarly, the large-scale process developed for the isolation of taxol by 
Polysciences Inc. from the bark of T. brevifolia consisted of the following steps, again 
starting with the alcoholic extract concentrate (Boettner et al. 1979). 

1 . Solvent partition water and CH2CL2, concentration to a solid 

2. Separation of the extract solid into soluble and insoluble fractions 

3. Chromatography on the soluble fraction 

4. Recovery of taxol and crystallization twice 

5. Silica chromatography on the taxol/ cephalomannine mixture 

6. Recovery and crystallization of taxol 

Thus, it appeared that, although procurement of taxol was of top priority, and 
many alternative approaches were attempted for solving this problem, one alternative, 
which was not considered, was to study the existing isolation procedure itself to make it 
more efficient. Thus the approach pursued at the University of Florida during 1990-91 
was to develop a simpler process for the isolation. Over the next few months, a new 
process was developed based on the use of a single reverse-phase chromatographic 
column, and consisting of the following steps, starting with the alcoholic extract 
concentrate (Rao, 1993). 

1 . Partition between water and chloroform, and concentration 

2. Reverse phase column chromatography on the extract directly 

3. Harvesting the crystals and recrystallization. 

The total chloroform extract of the bark of T. brevifolia, was applied directly to the 
CIS-bonded silica column in 25% acetonitrile in water (i.e., no separation into soluble 
and insoluble fractions); and the column developed with a step gradient (30-60% 
acetonitrile). The column fractions were let stand for 3-7 days, whereby taxol and seven 


of its analogues crystallized out directly from the fractions. These are filtered and 
purified further by recrystallization, or subjected to a small column. 

This process using reverse phase column chromatography, gave not only higher 
yields of taxol (0.02-0.04% vs. 0.01%) on a pilot plant scale, but also made possible the 
simultaneous isolation of a number of analogues which have not been obtained from this 
plant before. These included a 10-deacetyl baccatin III (0.02%); and a number of 
xyloside analogues, chief among which being the 10-deacetyltaxol-7-xyloside, which can 
now be isolated in yields of 0.1% or higher. 

Based on the successful fractionation of the bark extract of T. brevifolia, this 
technique was then ready for application to the other extracts such as the needles and 
wood of T. brevifolia, and to the needles of two other species of Taxus. These 
applications which gave practical methodology for processing these various extracts, 
also yielded many interesting taxoid compounds and these experiments are all detailed 
in this dissertation. 

Although this work was started during 1991 and much of the expected work was 
completed by late 1993, the world-wide interest in taxol research made a "quantum leap" 
at about this time, with a phenomenal increase in publications dealing with all aspects of 
taxol chemistry. Some of the compounds which were isolated for the first time in this 
laboratory, and whose structures were determined, were rediscovered by others and 
published. In spite of the enormous increase in the number of relevant publications, 
most of these publications described the isolation of the minimum possible amounts of 
the compounds, often as amorphous solids. Many determined their structures only 
through NMR spectral interpretation, with little or no other physical characterizations, 
elemental analyses, derivatizations or reactions. In at least a few examples, the 
assigned structures were found to be wrong and were subsequently corrected once or 

even twice. In the present work, practical isolation methods were used to obtain gram 
quantities of many compounds, as crystalline solids, where possible. 

The compounds are usually characterized by physical and spectral properties, 
providing elemental analyses, and carrying out derivatization such as acetylation, 
oxidation, etc. The structures were elucidated through chemical reactions as well as 
through spectral data. Thus, even though some of the final structures may have been 
published, the work described here contains experiments that have not been carried out 
by these authors. 

Brief descriptions of the topics that appear in this dissertation are given below. 

Chapter 2 gives a brief and selected summary of the pertinent literature on taxol 
and taxoids, covering the areas of isolation, elucidation of structures, semi-syntheses 
and total syntheses. Because the subject matter expanded enormously since 1993, the 
scope of the review is limited to material that is relevant to the subject matter of the 

Chapter 3 deals with the taxoid composition of the needles of Taxus brevifolia. It 
covers the application of the reverse phase column chromatography to the needle 
extract, isolation of the major taxoid, brevitaxane A (or brevifoliol), along with brevitaxane 
B, and taxol. It continues with the elucidation of the structure of brevitaxane A by 
various reactions, as well as by a detailed analysis of the NMR spectral evidence. 

Chapter 4 discusses some unusual reactions of brevifoliol. Such reactions have 
not been reported with this or any other taxoid compounds. In each case, crystalline 
compounds were obtained and characterized by physical and spectral data. 

Chapter 5 deals with fractionation of the extract of the needles of Taxus x media 
cv. Hicksii by reverse phase column chromatography and isolation of taxol and several 
other taxoids and their characterization. In spite of the fact that this species (ornamental 
yew) was declared as the preferred plant for the future isolation of taxol, no publications 


describing a suitable scheme for isolation of taxol or any other taxoids have appeared so 
far, other than analytical hpic data on their taxol content. 

Chapter 6 is similarly devoted to the fractionation of the extract of the needles of 
Taxus floridana Nutt. by reverse phase column chromatography. Isolation of taxol, 10- 
deacetyl baccatin III, baccatin VI and a new crystalline taxoid compound named 
taxiflorine, with its structural elucidation are described. 

Chapter 7 deals with the isolation of several crystalline non-taxane compounds 
present in the extracts of the bark and needles of Taxus brevifolia. These were shown 
to include flavonoids, phenols, and other types of compounds. 


An overview of the taxol story has been presented in Chapter 1 . In this Chapter, 
a selected review of the literature will be presented on taxol as well as the other taxanes, 
which are included in this dissertation. Two comprehensive reviews have been 
published on the subject of taxol, one by Kingston et al. (1 993) and one by Miller (1 980). 

The present review on the genus Taxus may be roughly divided into two parts: 
studies before and studies after the discovery of taxol. 

Earlier Studies 

The genus Taxus (N.O. Taxaceae) represents a group of plants (common name, 
yew) which grow mostly in temperate climates and can be found distributed throughout 
the world. They are generally slow-growing evergreen trees or shrubs with stiff linear 
leaves (or needles); and fruits which are small, fleshy and bright red. The common 
names of the plants are qualified by the place of its origin, as for example. Pacific or 
western yew {T. brevlfolia, Nutt.); European or English yew (7". baccata, Pilg.); Canadian 
yew (7. canadensis, Willd.); Japanese yew (7. cuspidata, Sieb. et Zucc); Chinese yew 
(7. chinensis, Pilg.); Himalayan yew (7. walllchiana, Zucc); ornamental yew (7 x media 
"Hicksii", Rehd.) and Florida yew (7 floridana, Nutt.). 

The toxic nature of this genus has been recognized for thousands of years, and 
in modern times, was first investigated chemically using the needles of Taxus baccata 
(Lucas, 1856). An amorphous mixture of alkaloids was isolated after extraction under 
acidic conditions and was given the name of "taxine." Further studies on taxine spanned 



many decades and covered many reactions relevant to taxane chemistry. In one sucli 
study, Winterstein and Guyer (1923) were the first to show the presence of 3- 
dimethylamino-3-phenylpropanoic acid in the hydrolyzate of taxine and this acid later 
became known as "Winterstein's acid." 

Until the 1960s most of the work on taxanes focused on these acid-extractable 
alkaloidal substances, which were readily separable from the large quantities of neutral, 
resinous materials which dominate the extract. Two groups of researchers were able to 
convert these somewhat unstable alkaloidal mixtures into more stable, non-basic 
substances in which the 3-dimethylamino-phenylpropanoic ester unit was transformed 
into a cinnamate ester. This, as well as the development of chromatographic 
techniques, made it possible to obtain pure compounds rather than mixtures. 

Baxter et al. (1958) in England investigated the major cinnamate ester obtained 
from T. baccata, which they named 5-0-cinnamoyl taxicin-l triacetate [2-1]. Similarly, a 
Japanese team (Nakanishi & Kurono, 1963; Kurono et al. 1963) studied a cinnamate 
ester from T. cuspidata, and called it 5-0-cinnamoyl taxicin II triacetate [2-2] and the 
structures of both these can be seen in Figure 2-1. These two compounds differ only at 
C-1, where taxicin II lacks the tertiary hydroxyl found in taxicin I. The lUPAC numbering 
system for taxanes used throughout this dissertation can also be seen [2-3]. 

A few years earlier, Graf & Betholdt (1957) succeeded in isolating the purified 
basic alkaloids, taxine A and taxine B from the original taxine mixture. Taxine B was 
shown to have the structure [2-4] (see Figure 2-1); which corresponded with 5-0- 
cinnamoyl taxicin 1 triacetate, into which it could be converted via elimination of the 
dimethylamine moiety. 


AcO OAc 


[2-1] - 0-Cinnamoyl Taxicin I Triacetate, R = OH [2-3]~IUPAC 
[2-2] - 0-Cinnamoyl Taxicin II Triacetate, R = H Numbering 



[2-4] - Taxine B 

[2-5] - Geranylgeranyl 

AD. 9 OH 





HO £ V ^^ 


[2-6a] - Proposed Glycol, [2-6b] - R = OAc, Baccatin II 

later corrected to 6 b ^2-7] - R = OH 10-DAB-lll 

Figure 2-1 : Early Studies on the Constituents of some Taxus Species 


Harrison (Harrison & Lythgoe 1966; Harrison et al. 1966) published one of the 
earliest biogenetic theories for the formation of taxanes starting with geranylgeranyl 
pyrophosphate and electrophilic cyclization [2-5]. Efforts by many groups to utilize a 
similar scheme to synthesize the taxane skeleton have been unsuccessful thus far 
(Kumagai et al. 1981; Hitchcock & Pattenden, 1992). Biogenetic pathways often provide 
ideas for simplified approaches in the synthesis of natural products. 

Many early studies utilized acidic conditions for the extraction which might have 
hampered the isolation of neutral or acid-labile compounds, Kondo & Takahishi (1925) 
obtained a non-basic compound from the Japanese yew by using neutral conditions. 
The cinnamates can also be directly isolated from the plant, indicating that they occur 
naturally and also as artifacts of processing. 

The National Cancer institute (NCI) and the U.S. Department of Agriculture 
(USDA) joined forces in 1960 to collect and screen plants for activity in several animal 
tumor models. Arthur Barclay of the USDA collection team obtained samples from the 
Pacific yew tree [Taxus brevlfolia Nutt., family Taxaceae) from Washington State in 
1962. In 1964, the extracts from the bark and stems were found to be active against KB 
cells in vitro (Wani et al. 1971). 

Studies after the Discovery of Taxol 

Dr. Monroe Wall had discovered another antitumor agent known as camptothecin 
using the activity on KB cells for isolation and was interested in any other extracts 
showing this activity. Thus, work on T. brevlfolia by Wani and Wall at the Research 
Triangle Institute was started and led to the isolation of 500 mg of taxol 2 years later in 
1966. Cytotoxic actions (Wani ef al. 1971) in KB cells, P388 leukemia, Walker 256 
carcinosarcoma and P-1534 leukemia were present in the extracts from the bark. These 


assays were all used at various points to monitor the fractionation, resulting in tine 
isolation of taxol as the active principle. 

In the 1960s the most straightforward and reliable method for the determination 
of complex chemical structures was x-ray diffraction analysis of a suitable crystal, also 
known as crystallography. Taxol crystallizes as thin needles not suitable for x-ray 
studies, but a tetraol derivative was amenable to x-ray studies. The structure of taxol [2- 
8] was determined by methanolysis (Figure 2-2); which yielded two compounds: the 
methyl ester of N-benzoyI phenylisoserine and an alcohol component shown to be a 
taxane tetraol [2-1 Oa]. This tetraol skeleton was converted into a 7,10-bis-iodoacetate 
derivative and, unlike all of the taxanes studied earlier, taxol showed the following 
unique features: 

1 . A taxane skeleton with an oxetane ring system involving C-4, C-5 & C-20 

2. An ester side chain consisting of N-benzoyI phenylisoserine at C-13 

3. A carbonyl function at C-9 

Baccatin III (Figure 2-1) and its 7a -epimer, baccatin V (Figure 2-2); were shown 
to be similar to taxol, having the oxetane ring and the C-9 carbonyl function. These 
epimers yielded better crystals and x-ray crystallography was performed. Baccatin III [2- 
6b] lacked the ester side chain present in taxol. Still another analogue, known as 10- 
deacetylbaccatin III (10-DAB, [2-7]) was later found to be much more widely distributed 
in Taxus spp., especially in the needles of Taxus baccata. This became an important 
taxane because it could be converted into baccatin III and later to taxol by the 
reattachment of the N-benzoyI phenylisoserine side chain at C-13 (See Figure 2-2). 

Taxol showed significant antitumor activity against a variety of in vivo murine 
tumors including B-16 melanoma and several human xenografts, which qualified it for 


RiO o 

[2-8] - Taxol, Ri = Ac R2 = CqH^ 
[2-9] - Taxotere, Ri= H R2 = OC(CH3)3 

RiQ O R2 R3 


HCT i AcO 


R1O OR2 

1 . . . 

HO" i AcO 


[2-10a] - Tetraol, R^ = R3 = H R2 = OH [2-11] - 7-0-TES 10-DAB III, Ri = H 

R2 = Triethyl Silyl (TES) 

[2-12] - 7-0-TES Baccatin III, Ri = Ac 
R2 = Triethyl Silyl (TES) 

[2- 10b] - Baccatin V, R-, =Ac 
R2 = H , R3 = OH 


[2-13] - 7-0-TES-13-0-Cinnamoyl Baccatin 

Figure 2-2 : Taxol and some Synthetic Targets 


clinical trials. A few studies on other species of Taxus tnave also been published, which r 

are referred to in Chapter 1 . 

Semi-synthesis of Taxol [ 

The relative ease in ester formation of the three hydroxy! groups in 10-deacetyl 
baccatin III (10-DAB, [2-7]) are 7>10»13. Esterification of the C-13 hydroxyl is very 
challenging due to the "inverted cup"-like folding of the taxane skeleton and strong 
hydrogen bonding with the carbonyl oxygen on the C-4 acetate. Before the side chain 
can be attached at C-13, the 7-hydroxyl must first be protected, often accomplished by 
attachment of a triethylsilyl group to give [2-11]. Next, this compound is acetylated at the 
10-position, to form 7-triethylsilyl baccatin III [2-12]. 

In one method, [2-12] was esterified with cinnamic acid to give [2-13], which was 
then converted to the phenyl isoserine ester by the Sharpless hydroxyamination 
procedure (Sharpless et al. 1991) using osmium tetroxide and t-butyl-N-chloro-N-sodio- 
carbamate (Mangatal ef al. 1989). The four isomers were separated and after 
deprotection of the hydroxycarbamates, N-benzoylation and deprotection of the 7- 
hydroxyl, taxol could be obtained. 

During the investigations of Greene and Potier (Denis et al. 1988; Kanazawa et 
al. 1994) dozens of side chain analogues were synthesized and tested, resulting in the 
discovery of the taxol analogue known as taxotere [2-9]. Taxotere® (docetaxel) was 
found to be more active than taxol in the tubulin assay and animal tumor systems and 
has also been approved as an antitumor agent. In an alternative synthesis, the 7- 
protected baccatin III [2-12] was esterified using either the chiral p-lactam [2-14] or the 
oxazinone [2-15] derivative to yield taxol. This method or some variation is currently 
used for the semi-synthesis of taxol and taxotere commercially from 10-deacetyl 
baccatin III (Ojima et al. 1991, 1992). 


Total Synthesis 

Swindell (1992) published a review on the progress of more than thirty groups 
and reported "only modest success" in the total synthesis of taxol. Only two years later 
two separate groups headed by K. C. Nicolaou (Nicolaou et al. 1994b) at the Scripps 
Research Institute and R. A. Holton (Holton et al. 1994b) at Florida State University 
would announce almost simultaneously two total syntheses of taxol. 

Nicolaou and colleagues designed the strategy for their synthesis based on the 
one bond disconnection analysis seen in Figure 2-3. After preparation of the fully 
functionalized A ring [2-16] and C ring [2-17] equivalents, a convergent and flexible 


McMurry coupling 




Acq ] O OH 

Shapiro reaction 


^Q formation 



[2-16] - Aryl sulphonylhydrazone 

[2-17] -Aldehyde 

Figure 2-3 : Nicolaou's Retrosynthetic Strategy 

synthesis of taxol involving 28 more steps allowed the preparation of numerous 
analogues. While not practical for the commercial production of taxol, synthetic methods 


provide researchers with a source of analogues for structure-activity relationships and 
lead to better methods of production in general. 

The first carbon-carbon bond between rings A and C was formed using a 
vinyllithium carbanion generated from the reaction of aryl sulphonylhydrazone [2-16], 
with n-butyl-lithium in tetrahydrofuran (THF); which was then combined with the 
aldehyde [2-17] in the Shapiro reaction (Shapiro, 1976) to produce [2-18] (Figure 2-3). 

Regioselective epoxidation of the A^'^'^-double bond was completed in 87% yield 
with f-butyl peroxide in the presence of V0(acac)2 leading to epoxide [2-19], which was 
then regioselectively opened with LiAIH4 to give the 1,2-diol [2-20] with a 76% yield. The 
carbonate introduced between the C-1 and C-2 hydroxyls in the next step served to 
position the two rings for ring closure and also allowed for the stereo-controlled 
introduction of the 2a-benzoate later in the sequence. The dialdehyde [2-21] needed for 
cyclization of the B ring was obtained after standard deprotection of the two primary 
hydroxyls and mild oxidation with tetra-/i-propylammonium perruthenate (TPAP) and 
4-methylmorpholine N-oxide (NMO) in acetonitrile. The previous three steps provided 
the carbonate dialdehyde in 32% overall yield. 

Formation of the B ring was accomplished with the versatile McMurry coupling 
(McMurry, 1989) under dilute conditions utilizing low valence titanium produced in situ 
from (TiCl3)2-(DME)3 (10 eq.) and Zn-Cu (20 eq.) in 1,2-dimethoxyethane (DME) at 70 ° 
C for 1 hour, giving the tricyclic A/B/C diol [2-22] with a 23% yield. 

Selective acetylation of the hydroxyl at C-10 rather than C-9 was expected due to 
allylic activation and proceeded with 95% yield. Mild oxidation of the C-10 hydroxyl was 
then carried out with TPAP-NMO in acetonitrile analogous to the oxidation to the 
dialdehyde with a 93% yield. 


After removal of the acetonide and protection of the primary hydroxy! at C-20 to 
make, the benzyl group was removed with catalytic hydrogenation and the 7-0-triethyl 
silyl protecting group was introduced to give [2-23]. Selective deacetylation of the 
primary acetate then provided the triol for the formation of the oxetane of ring D, which 
involves monotosylation at C-20 (primary OH) and triflate formation at C-5 (secondary 
OH) to produce [2-24]. Oxetane formation with a 60% yield occurs after mild acid 
treatment with catalytic camphorsulfonic acid (CSA) in methanol, followed by treatment 
with silica gel in dichloromethane. 

Acetylation of the C-4 position (tertiary hydroxyl) was followed by regioselective 
ring opening of the carbonate to the hydroxybenzoate functionality, both with good 
yields. The C-13a oxygen is introduced with pyridine chlorochromate in 75% yield 
followed by stereospecific reduction of the ketone [2-25] using NaBH4 in methanol in 
excess, for 83% yield. The hydroxyl is esterified using Ojima's p lactam synthon [2-14] 
(Figure 2-2) using the strong base sodium-hexamethyldisilazane for 87% yield based on 
90% conversion. Removal of the triethylsilyl groups with hydrogen fluoride in pyridine 
(HF-Pyr) completes the synthesis of taxol in 80% yield. 
Other Synthetic Approaches 

As previously mentioned Helton's group published a total synthesis of taxol in 
early 1994 at about the same time as Nicolaou, but their approach was quite different, 
with only a few reactions in common. Studies involving the fragmentation of bicyclic 
epoxy alcohols, referred to as "epoxy alcohol fragmentation," were the cornerstone of 
their syntheses of bicyclo[5.3.1] systems, including the unnatural epimer of (+)-taxusin 
[2-26], known as (-)-taxusin or enMaxusin [2-27] (Figure 2-6). 










Figure 2-4 : Nicolaou's Taxane Ring Synthesis 



HO PH oBn 






Figure 2-5 : Nicolaou's Final Synthetic Intermediates 


AcO OAc 



AcO OAc 



[2-26] - natural (+)-Taxusin 




AcO OAc 




H H 

Holton group - Patchouline Oxide Fragmentation 

[2-28] - Wieland-Miescher ketone 
Danishefsky group 


Wender group 

Figure 2-6 : Starting Points of Other Synthetic Strategies 


Danishefsky's total synthesis of baccatin III in 1996 (and hence, taxol); borrowed 
extensively from the experiences of Ojima, Holton, Nicolaou and others. The Weiland- 
Miescher ketone [2-28], available through catalytic asymmetric induction, allowed the 
installation of all stereochemical requirements to reach baccatin 111 in a sequential 
fashion. According to Danishefsky, "Our synthesis, though arduous, involves no relays, 
no resolutions, and no recourse to awkwardly available antipodes of the chiral pool" 
(Danishefsky ef a/. 1996). 

Wender's group published a most concise synthesis involving a-pinene [2-29] for 
construction of the ABC-tricyclic core of the taxanes (Wender & Rawlins 1992). Their 
approach takes advantage of the tendency for C-7 to undergo facile aldol/reverse aldol 
epimerization in taxol, allowing for aldol condensation under very mild conditions. 

General Structural Features of Taxanes 

The taxanes comprise a relatively large group of diterpenoid natural products 
covering a variety of structural patterns. These are believed to arise from geranyl 
geraniol [2-16], although the exact biosynthetic route has not been completely 
elucidated. A brief discussion of the major structural variations of taxanes is relevant to 
this work because many of these structures have been found in the compounds isolated 
in this work. A number of different forms that the C-20 diterpene skeleton itself can 
assume have been isolated. Next, the oxidation states, esterification patterns of the 
hydroxyls, and presence or absence of basic or neutral side chains allow for the 
extensive structural variation seen in these compounds. 

The taxane skeleton is a specific diterpene structure, consists of 20 carbon 
atoms arranged in a fused tricyclic system with the 6, 8 and 6 members in rings A, B and 
C, respectively. The double bonds at 1 1/12 and 4/20 are part of the basic ring system, 
although the latter may be modified by oxygenation to an epoxide or more commonly to 


an oxetane. As in the case of the analogous steroids with the two methyl groups as part 
of the skeleton, the taxanes have four methyl groups #16, 17, 18 and 19 as part of the 
taxane ring system. Some examples of the taxane skeleton found in the various species 
of Taxus are shown in Figures 2-1 and 2-2. 

Oxygenation of the taxane ring has been observed to varying extents. The 
minimum number being 4, distributed at 5, 9, 10 and 13, as seen in taxusin [2-26]. In 
general, oxygenation may occur at carbons 1, 2, 4, 5, 7, 9, 10 and 13. Instances have 
been recorded where oxygenation was present at 14 (in place of 13), as well as part of 
the methyl groups at 19 and 17. 

This is the most common structural type seen in the taxanes, with a C-4(20) and 
a A^^ double bond. These taxanes are generally referred to taxa-4(20);11-dienes. The 
alkaloidal Winterstein esters are included in this group as are many of the neutral 
taxanes. The oxygen at C-9, if present, is usually seen as a secondary alcohol or as an 
ester. The C-13 position in this group, likewise, exists as an alcohol, ester or oxidized to 
a carbonyl to form an a,|3-unsaturated carbonyl. Esterification at C-13 is usually limited 
to an acetyl or a cinnamoyi, but the side chain (N-acyl phenyl isoserine); as found in 
taxol, cephalomannine and others has not been reported in this subgroup so far. The 5 
position is oxygenated with an a-hydroxyl, which might be free, or esterified by an acetic 
acid, cinnamic acid or the Winterstein-acid. Some examples of these compounds with a 
cinnamate ester function are described in Chapter 5. 

This group is relatively less frequent but examples with different substitution 
patterns have been isolated. One variation comes from the presence or absence of 
hydroxyl at C-1. Members of this subgroup also generally contain the 5-a-hydroxyl, 


which is estehfied in the same fashion as the dienes above to provide further variation. 
An unusual example is the taxane with the C-9-nicotinoyl ester function, found in 
Austrotaxus spicata Compton Taxaceae (Ettouati e^ al. 1988). 

This group is characterized by having an oxetane ring system involving the 
carbons 4, 5 and 20. It may be divided into two subgroups based on whether they 
contain the phenyl isoserine ester side-chain at C-13 or not. The former contains taxol 
and all of the other compounds, which are active in the tubulin assay and hence are of 
much importance. Division into two other subgroups is also possible in those without the 
C-13 side chain, with one having a carbonyl at C-9 and with a hydroxyl or an esterified 
hydroxyl at C-9. 

The oxetane-containing taxanes are generally highly oxygenated and often have 
oxygen at C-1, 2, 4, 5, 7, 9, 10, and 13. In some special instances, a hydroxyl has been 
reported at C-1 9 (Fuji et al. 1993). The phenylisoserine ester side chain has been seen 
in the form of at least three different amides that occur in nature. These are taxol, with 
the N-benzoyI group, cephalomannine, with the N-tiglioyI group and taxol C, with the N- 
hexanoyl group. 

Taxol has a complex structure and knowing what features of this structure are 
necessary for the activity is of utmost importance and this aspect has been studied using 
the in vitro tubulin binding, and the cell culture assays and a summary of these data is 
presented below (Samaranayake et al. 1993). 

Acylation of the 2' position of taxol does not destroy cytotoxicity but does stop 
promotion of microtubule assembly. Bulky acyl groups reduce the activity in the cell 
culture, thus suggesting that hydrolysis of the 2' position back to a free hydroxyl might be 


Substitution of the 7 position does not appear to significantly decrease the 
activity. Taxanes with a 7p-0-xyloside moiety are comparably active in both assays 
when compared to the respective aglycones. Similarly, epimerization at the 7-position 
does not eliminate activity. 

Hydrolysis of the 10-acyl function does not reduce the cytotoxicity significantly in 
cell culture assays. As with other structural features, this point is being explored in the 
more recent clinical trials in Europe with taxotere. 

The importance of the oxetane ring for activity has been investigated through ring 
opening via different Lewis acids including Meerwein's reagent (triethyloxonium 
tetrafluoroborate); acetyl chloride, mesyl chloride and others. The product obtained form 
the Meerwein's reagent had a primary alcohol at C-20 and secondary C-5-hydroxyl, but 
no other changes compared to taxol. The activity normally seen with taxol in both 
assays was lost with the opening of the oxetane hng. This suggests that the oxetane 
ring is necessary for activity but leaves open questions regarding the effect of ring 
contractions in ring A. 

The properties of the C-13 hydroxyl mentioned above make attachment of a side 
chain quite difficult. Protection of other free hydroxyls in both the side chain and taxane 
skeleton are necessary, followed by selective deprotection after the side chain has been 
attached. Taxotere and taxol have both been synthesized from this taxane and this is 
currently the starting material for the production of both drugs. 

Epimerization of the 7 hydroxyl from p to a via a retro-aldol mechanism allows 
formation of an energetically favorable hydrogen bond with the 4-acetate carbonyl 
oxygen. This epimerization is a concern in both taxane isolation and synthetic methods, 
and necessitates the avoidance of acidic or basic conditions. Protection of this C-7 p- 


hydroxyl with groups such as a chloroacetate avoids both epimerization and unwanted 

reaction at this position. 


A number of taxanes in which the A-ring is isomerized to a 5-membered ring to 
give a 5/7/6 instead of the 6/8/6 system have been isolated and these are termed 
abeotaxanes. They are again divided into two groups into a) those with the 4/20 
unsaturation and b) those with an oxetane ring at this location. We isolated the first 
members of each of these groups in our work, e.g. brevifoliol (Chapter 3); and the 
compounds isolated from the bark of T brevifolia described in Chapter 6. As indicated 
earlier, treatment of taxol with acidic reagents can isomerize ring A to form such 
compounds, although these compounds are naturally present in the extract and not 


Taxol was originally isolated from the bark of the Pacific yew {Taxus brevlfolia 
Nutt., N.O. Taxaceae). As indicated in Chapter 1, during 1991-1993 there was a 
reassessment of the use of the bark as the source. This concern resulted in an intense 
search for alternative sources for taxol that are renewable, with sources such as the 
needles of the yew tree instead of the bark. This laboratory was also involved in this 
search and looked into the needles of three different yew species as a source for taxol.' 
T. brevifolia, T. x media Hicksii and T. floridana. The taxane composition of T. brevifolia 
needles is the subject of this chapter. I 

Fractionation of the Needles of Taxus brevifolia i 

A quantity of 100 lbs. of the needles of T. brevifolia was obtained from a supplier 
in Oregon. They were air-dried and extracted with methanol at room temperature and 
the extract was concentrated under reduced pressure to a syrup. This was partitioned 
between water and chloroform, and the organic layer concentrated to give a dark 
greenish brown semi-solid, called "extract solids", which represented about 5% of the dry 
weight of the needles. 

It was decided to follow the method successfully developed with the bark extract 
for the fractionation of the extract solids, using preparative scale, reverse phase column 
chromatography. Direct application of the crude chloroform extract of the needles onto a 
C-18 bonded reverse phase silica column was accomplished as described in the 
experimental section. After placing the extract-containing silica onto a 25% acetonitrile 



in water column (1:4 ratio of loaded to clean silica); a step gradient of acetonitrile in [ 

water mixtures was performed up to 60% acetonitrile. I 

Preliminary studies on the extract solids of the needles by TLC and analytical | 

HPLC showed that the sample contained somewhat minor amounts of taxol. A i 

predominant component that was slower moving than taxol in TLC gave a greenish-blue 
colored spot when sprayed with 1 N sulfuric acid and heated on a hot plate (charring). 
Likewise, in the analytical HPLC, this component appeared after 10-deacetyl baccatin III 
as the major constituent judging from the peak heights, but before taxol and at least 
several times more abundant. 

The reverse phase column (C-18 bonded silica gel) on the needle extract 
concentrate was started with 25% acetonitrile in water. The sample was carefully 
prepared as a slurry (see experimental) and added to the column. The column was 
developed using a step gradient of acetonitrile in water 30-60%. Fractions of suitable 
volume were collected and monitored by absorbance at 275 nm, TLC and analytical 
HPLC. Four regions were recognized in the elution profile of the column, based on the 
UV absorbance (275 nm.); which contained the resolved constituents of the extract. 

The early fractions contained components, which accounted for the bulk of the 
UV absorbance of the sample. These appeared to be non-taxane phenolic compounds 
with or without attached sugars. A description of these will be given in Chapter 7. The 
first taxane component, which appeared at the 35-40% acetonitrile elution, was also the 
major component. It was collected from the appropriate fractions, and after 
concentration, obtained as a crystalline solid. Next, fractions from the 50% acetonitrile [ 

elution contained taxol, which was obtained as a crystalline solid directly from the i 

fractions. Following this, the fractions from the 55-60% acetonitrile elution gave another , 

taxane component which gave a greenish blue spot on the TLC (after charring with 
sulfuric acid) similar to the major constituent referred to above. i 

Brevitaxane A (Brevifoliol) [3-11 

The major constituent, which was obtained in a yield of 0.2-0.25%, was named 
brevitaxane A because the physical and spectral data indicated that it was a new taxane 
compound (later renamed by others as brevifoliol, which will be used throughout this 
dissertation). Elemental and FAB-MS analysis (MH+ 557) agreed with the molecular 
formula of C3iH4o09(Balza et al. 1991). 

An examination of the ^H NMR spectrum showed the presence of two acetyl 
groups (signals at 5 1.76 and 5 2.07); and a benzoate group {5 7.88 (d); 5 7.43 (t) and 5 
7.56 (t)}. The spectrum also gave evidence for the presence of a (4/20) exocyclic double 
bond (two characteristic broad singlets at 5 4.82 (H-20A) and 5 5.20 (H-20B) and signals 
at 5 1 12.1 (C-20) and 5 149.0 (C-4) in the ^^C NMR spectrum. 

Very little information on the various types of taxane structures that are known 
now was available at that point in time (1991) and even less on their diagnostic spectral 
characteristics. Based on analogous taxanes and the evidence outlined above it was 
postulated that this major constituent had the relatively common 4/20,1 1-taxadiene type 
skeleton. The presence of an exocyclic 4/20 double bond and absence of an oxetane 
ring supported our initial assumptions. The next step was to determine the positions of 
the various substituents in the molecule in order to elucidate the complete structure. 

Most of the structural elucidations of taxanes at the time were based on 
degradative studies. It was decided to follow this lead in establishing the presence of 
the various functionalities as well as their location in brevifoliol, by actual reactions 
and/or derivatizations, supplemented by spectral methods. 

















Hydroxyl Functionalities 

i) Acetylation: To determine the number and positions of all hydroxyls in the 
molecule, the compound was subjected to acetylation. Two products were obtained 
under mild conditions (20 °C, 15 min). These two were separated by chromatography 
and both obtained as crystalline solids. One was shown to be a monoacetate and the 
other a diacetate. 

Table 3-1 : Proton NMR Spectra of Brevifoliol and Brevifoliol Acetates 



(J in Hz) 



5 -Ac 












1.49 cm 

1 .46 cm 

1 .47 cm 

1.46 brd (13) 


2.36 dd (9,13) 

2.40 dd(9, 13) 


2.41 dd(9, 13) 

2.65 dd(9. 


2.78 d (9) 

2.76 brd (9) 

2.91 d (9) 

2.72 br d (9) 

2.71 brd (9) 


4.45 br s 


4.37 brs 

5.39 brs 

5.38 dd (4, 2) 


1.86 cm 

1.88 cm 

1.85 cm 

1.90 cm 

1.87 cm 

2.02 cm 

2.0 cm 

1.99 cm 

2.00 cm 

2.0 cm 


5.56 dd (5,11) 

5.62 dd (5, 11) 


5.61 dd (5, 

5.63 dd(5. 


6.05 br 


6.07 d 

6.09 br 

5.8 d (10.8) 


6.53 d (10.6) 

6.63 d (10.6) 

6.66 d 

6.65 d (10.6) 

6.64 d (10.8) 


4.38 t (7.5) 

4.53 brt (7.2) 

5.46 br s 

5.54 brt (7.2) 

5.61 t(6.9) 



1.22 dd* 

1.32 cm 

1.25 dd* 

1.25 dd* 


2.42 dd * 

2.51 cm 

2.51 dd* 

2.62 dd * 


1.05 s 

1.03 s 

1.09 s 

1.11 s 

1.63 s 


1.35 s 

1.33 s 

1.35 s 

1.35 s 

1.71 s 


2.01 s 

2.06 s 

2.02 s 

2.03 s 

1.96 s 


0.90 s 

0.91 s 

0.89 brs 

0.92 s 

0.92 s 

20 A 

4.82 brs 

4.90 brs 

4.80 brs 

4.92 brs 

4.89 brs 

20 B 

5.20 brs 

5.28 brs 


5.28 brs 

5.29 brs 


7.88 d( 7.5) 

7.87 d (7.5) 

7.87 d (7.5) 

7.87 d (7.5) 

7.84 d (7.5) 


7,43 t( 7.5) 

7.43 t (7.5) 

7.44 t (7.5) 

7.44 t (7.5) 

7.42 t( 7.5) 


7.56 t( 7.5) 

7.55 t (7.5) 

7.56 t (7.5) 

7.56 t (7.5) 

7.53 t( 7.5) 


1.76 s 

1.76 s 

1.76 s 

1.75 s 

1.77 s 

2.07 s 

2 06 s 

2.05 s 

2.02 s, 2.07 s 

2.02 s, 2.08 s 


2.06 s 

2.08 s 

2.09 s, 2.11 s 

NMR were recorded at 600 MHz in CDCbon a Varian Unity 600 instrument at 
ambient temperature. Chemical shifts 5 (ppm) are reported with TMS as internal 


Table 3-2 : 

Carbon NMR Spectra of Brevifoliol and Brevifoliol Acetates 





































































































































































169.9 (X2) 




^^C NMR spectra were recorded at 150 MHz in CDCIson a Varian Unity 600 
spectrometer at ambient temperature. Chemical shifts 5 (ppm) are reported with TMS as 
internal standard. 



BzO OAc 

[3-6] - 13- Ketone 

F^1 ^2 F^3 

[3-1] - H H H 

[3-2] - Ac H H 

[3-3] - H Ac H 

[3-4] - Ac Ac H 

[3-5] - Ac Ac Ac 




OH ^3C 

[3-7] - 4, 20- D hydro 



[3-8] - 4,20-Epoxide 




[3-9] - Norketone 






[3-1 1] - Hydrolysate, Ri = R2 = H 
[3-1 5] - DebenzoyI, Ri = R2 = Ac 

Figure 3-2 : Brevifoliol and Reaction Products 


Appropriate conditions under which each of these could be obtained as exclusive 
products were developed. At room temperature in acetic anhydride for 1-2 minutes 
before quenching the reaction, the monoacetate was the major product (>90%). 
Likewise, at 80 ° C for 30 min. the product was the diacetate. 

The ^H NMR spectral data for the monoacetate showed that the signal at 5 4.45 
(br s) shifted to 5 5.37 (dd, J=4.2, 2.4 Hz); indicating that acetylation took place at the 5- 
OH, as shown in [3-2]. In the diacetate, besides this shift for the 5-OAc, the signal at 5 
4.38 (t, 7.5 Hz) shifted to 5 5.54 (br t, 7.2 Hz); thus showing that the second acetate was 
located at C-13 [3-4]. A naturally occurring brevifoliol 13-acetate [3-3] was isolated and 
1,5,13-brevifoliol triacetate [3-5] produced in this lab will be discussed in Chapter 4. 

ii) Oxidation: Brevifoliol was readily oxidized by manganese dioxide (MnOa) in 
refluxing benzene to yield a ketone product. In the ''H NMR spectrum, a major change 
was the absence of the triplet at 5 4.38 due to the C-13 proton, thus showing that the 
oxidation took place at the 13-OH [3-6]. Further evidence was seen by the shift of the 
signals for the C-14 protons from their normal positions at 8 1 .29 (dd, 14.0, 7.6 Hz) and 5 
2.46 (dd, 14.0, 7.6 Hz) to 5 2.32 (d, 19 Hz, H-14a) and 5 2.48 (d, 19 Hz, H-14(3). When 
brevifoliol was oxidized by Jones reagent, the same 13-keto brevifoliol seen with Mn02 
initially formed [3-6], With time the initial product gradually disappeared, giving rise to a 
faster moving product. This second oxidation product was shown to be the result of an 
unusual reaction described in Chapter 4. 

4/20 Unsaturation 

i) Hydrogenation: When hydrogenated in the presence of 5% Pd/carbon, 
brevifoliol gave the dihydro derivative [3-7]. In its ^H NMR spectrum, the characteristic 
signals at 5 4.82 and 5 5.20 due to the C-20 protons were absent and a new methyl 


doublet and a new methine proton appeared. In the "C NMR spectrum the 
characteristic signals from the exocyclic 4/20 double bond were absent, accompanied by 
the appearance of new methyl and methine signals. 

ii) Epoxidation: Brevifoliol was heated in dichloromethane with meta-chloro 
peroxybenzoic acid (MCPBA); whereby it underwent oxidation to yield the epoxide [3-8], 
a crystalline compound. 

iii) Ozonization: Brevifoliol has two double bonds, one at the 11/12 position and 
the other at the 4/20 position. Of these, the former is tetra-substituted, while the latter is 
of an exocyclic methylene type. No information was available in the literature regarding 
the reactivity of the taxane skeleton to indicate whether one or both double bonds would 
be cleaved by ozonolysis. In the present work, ozonization was carried out in a mixture 
of methanol and dichloromethane -70 ° C. After the disappearance of the starting 
material, the ozonide was decomposed with dimethyl sulfide and the products isolated 
by chromatography. Two major products were separated. The first was the same as the 
epoxide [3-8] obtained by reaction with MCPBA. The second was the expected 
ozonolysis product in which the 4/20 double bond was cleaved to form the ketone [3-9]. 

iv) Formation of a did: As one of the characteristic reactions of an ethylenic 
function, oxidation by osmium tetroxide was attempted with brevifoliol. The reaction 
proceeded smoothly to give a diol [3-10]. 

Number and Nature of the Oxygen Substitution 

From the preceding discussion it is evident that brevifoliol has two free hydroxyls, 
two acetoxyls and one benzoyloxy functions. However, in the "C NMR spectrum of 
brevifoliol, the number of oxygen substituted carbons was six; 5 70.1, 8 70.2, 5 72.4, 5 
75.9, 5 76.7 and 5 77.2. To determine if one of the six is a different type of an ester, or a 
tertiary hydroxyl, brevifoliol was subjected to saponification in alcoholic KOH to yield the 


hexaol [3-11], obtained as a crystalline solid. This was then acetylated to a crystalline 
acetate [3-12]. The ^H- and ^^C NMR spectra of [3-12] showed the presence of 5 
acetates (^H: 5 21.8, 5 21.7, 5 21.4, 6 21.3, 6 21.0, 5 20,8; and "C 5 171.0, 5 170.4, 5 
169.8, 5 169.6 and 5 169.5); which suggested that brevifoliol contained a tertiary 

hydroxyl. ' 

In the conventional taxane skeleton, a tertiary hydroxyl is often present at C-1, ^ 


with the other hydroxyls (or esters) at C-2, C-5, C-7, C-9, C-10 and C-1 3. Thus, with | 

brevifoliol having five oxygen substituents, one of these positions must be without | 

attached oxygen. Thus, it would be important to know which of these positions does not 
have an oxygen substituent. For this reason, the hexaol [3-11] was subjected to 
oxidation by periodate. If there were two pairs of vicinal hydroxyls, e.g. 1,2 and 9,10, the 
hexaol will be cleaved in such a way as to give smaller molecules which represent the A 
and C rings. If there is only one such pair, the reaction will produce a product with all of 
its carbons intact. The hexaol undenwent oxidation readily to form a dialdehyde [3-13] 
without losing any carbon atoms found in original carbon skeleton. Unaware of the 
unusual A ring structure, it was presumed that the presence of a tertiary hydroxyl at C-1 
precluded the presence of oxygen substitution at C-2. Additional evidence for a 
methylene carbon at C-2 was found in the COSY spectrum from the chemical shifts in 
the H-3p-H-2a-H-2p isolated spin system. 

Thus, brevifoliol has two hydroxyls at 5 and 13. Locating the benzoate group at 
one of the three choices, 7, 9, or 10 will elucidate the structure. At this point, brevifoliol 
was required in microbial and fungal biotransformation project in our laboratory. In order 
to produce an antiseptic sample an aqueous alcoholic solution was sterilized in a steam 
autoclave at 125 ° C, 20 atm., to see if it is stable. It was found that the compound 
underwent degradation to give two or three products. 








[3-12] - Ftentaacetate f rom Hexaol 

[3-13] - Fteriodate Oxidation 

[3-14] - Fteriodate Oxidation Osazone Product 

Figure 3-3 : Brevifolio! Hexaol Reaction Products 

The major component of this mixture was found to be debenzoyi brevifoliol 
(Figure 3-1 [3-15]). Of the three possible locations, 7, 9, and 10 for the benzoate, only 
10 is allylic and hence the ester at this position is more likely to be labile. Taxol with the 
benzoate at C-2 is completely stable to heat and pressure for hours. This evidence, 
along with chemical shift arguments concerning the effect of acetylation versus 
benzoylation, led us to place the benzoate at C-10. 

This group presented the isolation and the structural elucidation of brevitaxane A 
at the International Research Congress on Natural Products held in Chicago, IL in July 


1 991 . Balza e^ al. (1 991 ) published the isolation of a new compound at about that same 
time from the needles of T. brevifolia, which they named brevifoliol, and an assignment 
of its structure as shown in [3-16]. The compound appeared to be similar to, if not the 
same as, brevitaxane A, that was isolated from the needles at the University of Florida. 
The structure proposed by Balza et al. differed from that of brevitaxane A, with the 
benzoate group being placed at C-7 instead of at C-10. 

That same year (1992); the isolation of taxchinin A was described (Fuji et al. 
1992); which was later shown to be 2-acetoxy-brevifoliol. Fuji correctly assigned the 
structure with a 5-membered ring A, on the basis of NMR spectral data. The authors who 
isolated brevifoliol and assigned structure with the 7-benzoate (Chu et al. 1993) 
published a revised structure for brevifoliol, in which the benzoate was moved to C-10, 
from C-7, but with the skeleton of a conventional taxane. 

During 1993, two other publications appeared, one from Georg et al. (1993). 
and the other from Appendino et al. (1993) reexamining the NMR spectral data of 
brevifoliol, and arriving at the structure in which the A-ring was 5-membered. Later that 
year, Chu et al. (1993); on the basis of x-ray crystallographic data, revised the structure 
of brevifoliol again to the presently accepted structure. 

Due to the intense competition in "taxol research", we began a detailed 
examination and analysis of the NMR spectral data using the ^^C NMR, NOESY, 
HETCOR and other spectral methods to determine if the rearranged (5/7/6) skeleton 
might be distinguishable from the spectrum of a taxane with a conventional (6/8/6) 
skeleton. The following is an analysis of the spectral data of brevifoliol. 

The carbonyl signal in the ^^C NMR spectrum at 5 164.3 indicated the presence 
of one benzoate, and signals at 6 169.9, 5 170.5, likewise, indicated that 2 acetate ester 
groups were present. Further support for the benzoate was obtained by the four 


aromatic signals between 5 128.7 and 5 133.2 (see tables 3-1 and 3-2); and for the two 
acetates, by the methyl signals at 5 20.7 and 5 21.4. Analysis of the ^H NMR and ^H 
COSY and ^H.^^C Heteronuclear Correlation (HETCOR) experiments also gave 
additional support for the presence of the acetates with signals at 5 1 .76 s and 5 2.07 s, 
as well as benzoate signals at 5 7.88 d (ortho); 5 7.43 t (meta); and 5 7.56 t (para). Next, 
evidence for the presence of the normally present (11/12) taxane double bond could be 
seen in the carbon spectrum by the signals at 6 133.9 (C-11) and 5 151.5 (C-12);. 
Similarly, the existence of a (4/20) exocyclic double bond could be seen by the signal 5 
149.0 (C-4) and 5 112.1 for (C-20). In the ^H NMR spectrum the exocyclic 4/20 double 
bond is also indicated by the two characteristic broad singlets seen at 5 4.82 (H-20A) 
and 5 5.20 (H-20B). 

In the ^H COSY experiment weak but definite interactions between the singlet at 
5 4.82 (C-20a) with both the H-3p doublet at 5 2.78 (9 Hz.) and the H-2a doublet of 
doublets at 5 2.36 (9, 13 Hz.) supported the assignments given for the methylene 
protons. (The designations for the C-20 protons are A and B, since a and p do not have 
the conventional meaning system and could be confusing). Along with the interaction 
between H-2a and H-2p the first isolated spin system in the ^H spectrum was 
established and the relative geometry of the protons. 

The region between S 62.4 and 5 77.1 in the ^^C spectrum carbons with hydroxyl 
or ester oxygen attached to oxygens, and these signals could be further defined in the 
DEPT experiment (Distortionless Enhancement with Polarization Transfer, NMR) as 
primary, secondary, tertiary and quaternary carbons. The spectrum showed two 
quaternary carbon signals and five oxymethine carbon signals. Since the presence of 
only six signals was expected based on the proposed formula, the quaternary signal at 
5 62.4 was intriguing even from the start of the spectral examination. In taxol with its C-9 


carbonyl, the C-8 signal appears near 5 58, so the signal at 8 62.4 immediately raised 
questions about the true structure of brevifoliol. This signal did not fit the normal 
chemical shift pattern of any naturally occurring taxanes known at that time. In the 
absence of a carbonyl group at C-9, the C-8 carbon usually falls in the region of 5 40-50 

Unable to satisfactorily explain this unusual peak position, the Chemistry 
Department was contacted about crystallographic services. X-ray crystallographic 
analysis was performed by Dr. K. A. Abboud on the 5-monoacetate [3-2]. Surprisingly, 
the presence of an unusual 5/7/6 ring system was evident, where the normal 6- 
membered A ring of the conventional taxane system was "rearranged" to form a 5- 
membered ring with the carbons 15, 16 and 17 moved out of the ring system to form a 
hydroxy isopropyl group at C-1. Since the x-ray structure was obtained on brevifoliol-5- 
acetate, it was important to establish whether brevifoliol itself had this rearranged taxane 
skeleton, or if the rearrangement could have occurred during the acetylation. 

This structure represented a departure from the existing naturally occurring 
taxane structures available at that time, previously seen only as a product of 
rearrangement under strongly acidic conditions (Samaranayake et al. 1990). 
Crystallography of the original compound was not done because it failed to yield 
adequate crystals for analysis without prior acetylation. This made it necessary to 
determine whether this new ring structure was naturally occurring, or formed during the 

In one such ring contraction, taxol underwent rearrangement of the A-ring, 
accompanied by dehydration, to produce an isopropenyl group at C-1, as well as other 
changes such as the opening of the oxetane ring. Since the "C NMR spectra of both 
brevifoliol and its monoacetate showed these signals at 6 62.4 assigned to the C-1 


carbon and the one at 5 75.9 assigned to the quaternary C-15 containing tertiary 
hydroxyl, it appeared unlikely that such a rearrangement took place during the 
acetylation. HETCOR and APT experiments corroborated these conclusions, thereby 
agreeing with the structure determined by the x-ray crystallographic method. 

Further analysis of the ^H COSY spectrum revealed an isolated spin system of 
two doublets due to H-9p at 5 6.05 and H-10a at 5 6.53, with a pseudo-axial orientation 
indicated by the degree of splitting (J=10.6 Hz); and significant broadening of the signal 
at 5 6.05. Some amount of the deshielding of H-10a relative to H-9p was expected, due 
to the adjacent double bond, which makes the C-10 position allylic. The presence of a 
benzoate at this position would be expected to cause a further downfield shift based on 
analogous compounds already known (Chu et al. 1992). With a thorough analysis of the 
■■h NMR and ^H COSY spectra, the signal at 5 4.38 (t, 7.6 Hz) was assigned to the H- 
13p proton, which coupled strongly with H-14P at 5 2.46 (dd, 14.0,7.6 Hz.); as well with 
H-14a at 5 1.25 (dd, 14.0, 7.6 Hz). Weak long range coupling to the C-18 methyl 
protons at 8 2.01 was also evident, as the slight broadening of this peak is generally 
attributed to this long range coupling in other taxanes. 

The isolated spin system of H-5p, H-6a, H-6p and H-7a is easily identified in 
most taxanes, with a tendency to show a sharp multiplet for H-7a and broader, poorly 
resolved splitting for H-5p, especially if H-5p is not esterified (Delia Casa de Marcano & 
Halsall, 1970; Rao et al. 1995). The H-5p broad singlet at 5 4.45 interacts with the H- 
6a multiplet at 5 1.86, which interacts with the H-6p multiplet at 5 2.02, which in turn 
interacts with the H-7a signal at 5 5.56 (dd, 5,1 1 Hz.). In many cases esterification of a 
hydroxyl causes a deshielding effect on the related proton of about 1 ppm. The 
chemical shifts and splitting patterns indicated that the acetate groups were at C-7 and 
C-9, with the benzoate at C-10. 


The remaining carbons are the 4 methyl groups usually seen in taxanes on C-15 
(methyl 16 and methyl 17); at C-12 (methyl 18) and at C-8 (methyl 19). The methyl 
group located on the 11/12 double bond (methyl 18) is often quite deshielded in the 
proton spectrum (5 2.01, s) but shielded in the carbon spectrum (5 12.0). This usually 
aids in its assignment along with further evidence from Heteronuclear NMR experiments. 
Methyl 19 is usually shielded in both the ^H (S0.90, s) and "C (5 12.9, q) spectra, as 
seen here. 

This class of compounds commonly referred to as 11(1-»15)-at)eo-taxanes or 
occasionally A-nortaxanes. Many compounds of this type are now known, some 
containing the 4/20 unsaturation as in brevifoliol and others with a 4/20 oxetane structure 
as seen in 1 1(1~>15) abeo baccatin VI. 


Extraction of the Needles of Taxus brevifolia 

The needles obtained from a supplier (Mr. Patrick Connolly, Yew Wood 
Industries, 6928 North Interstate Avenue, Portland, OR 97217) were air-dried for one 
week. The dried needles (20 Kg) were extracted by immersing in methanol at room 
temperature. After two days, the extract was drained, concentrated under reduced 
pressure at temperatures below 35 ° C. The recovered methanol was reused for a 
second extraction, which was processed the same way. After two more extractions, the 
combined concentrate was freed from some more of the methanol to obtain a dark green 

The above syrup was partitioned between water (10 gallons) and chloroform (10 
gallons). The organic layer was separated and the extraction carried out twice more 


using 5 and 3 gallons respectively. The combined chloroform extract was concentrated 
under reduced pressure to reach a dark green semi-solid stage (800-900g). 
Reverse Phase Column Chromatography: 

The column used was a threaded glass column of the Mitchell-Miller type (2.5 x 
24") with the appropriate fittings, purchased from Ace Glass Co., Vineland, NJ suitable 
for low pressure liquid chromatography. A slurry of the C-18 bonded silica (800 g) 
(Spherisorb, 15-35 micron diameter) purchased from. Phase Separations Inc., Norwalk, 
CT) in methanol was poured into the column, which was run under a gentle pressure by 
using a metering pump (Fisher/Eldex) until an adequately packed bed was obtained. 
The column was then equilibrated with 25% acetonitrile in water, to prepare for the 
addition of the sample. 

The extract solids (200 g) was dissolved in acetonitrile (400 ml) by warming to 
make sure that no lumps remained. To this was added approximately 200 g equivalent 
of the equilibrated resin (about 20% of the column packing) with stirring. As the stirring 
continued, the slurry was diluted with 25% acetonitrile in water (500 ml); followed by 
water to make up a total volume of approximately 2 L. The stirring was continued with 
occasional warming to 50-60 ° C for about 15 min. At this point, a sample of the slurry 
taken into a test tube, showed that the silica settled readily to give a clear supernatant 
and no green precipitate or oily material was present. The slurry was then filtered using 
light suction and the solid (silica with the sample) re-slurried using part of the filtrate and 
the thick slurry added to the column. The rest of the clear supernatant was then pumped 
on to the top of the column using the metering pump. From time to time, the column 
feed was checked to see that it remained clear, and if not, to either warm briefly or add 
minimal amounts of acetonitrile to it until it became clear, so as to prevent precipitate 
from appearing and blocking the pump. 


After the sample addition was completed, fresh 25% acetonitrile/ water was 
passed through, followed by the step gradient of acetonitrile/ water (30, 35, 40, 45, 50 
and 60%) was used. Fractions (200 ml) were collected and monitored by UV 
absorbance (at 275 nm), TLC and analytical HPLC. The change to the next 
concentration of solvent was determined by the results of monitoring the fractions. For 
example, when the absorbance values rose as a result of the previous change, the 
solvent was continued until a definite trend to lower values was seen. Similarly, when 
the TLC showed the trend towards decreasing intensity of the major spot, and no new 
spot had shown a tendency to increase, the solvent was changed to the next level. In 
general, 2-3 multiples of the hold-up volumes of the column were used. 

After the elution with the 60% solvent was completed, the column was washed 
with 100% methanol, followed by a mixture of methanol/ ethyl acetate/ ligroin which 
stripped the column of the chlorophylls, waxes and other lipid soluble components. After 
this solvent, washing with methanol and equilibration with 25% acetonitrile/ water made 
the column ready for another run. 

After the monitoring, fractions with low UV-absorbance values were combined 
and concentrated into groups, based on the TLC data. Those fractions with relatively 
stronger UV readings were let stand at room temperature for 3-5 days, whereby crystals 
appeared in several sections of the fraction sequence. These crude crystals were 
filtered, dried and purified further either by recrystallization or using a small silica column 
(normal phase). 

Brevifoliol [3-11 

The fractions containing this component gave crystals but only a small portion 
was obtained in this form. Hence, after filtration of the crude crystals, the filtrate was 
concentrated to dryness and the solid taken up in dichloromethane and passed through 


a column of normal phase silica, using a ratio of 3-5 g of silica per gram of the solid. The 
effluent and washes which contained the compound were combined, concentrated to 
dryness and the solid crystallized from a mixture of acetone and ligroin to obtain 
brevifoliol as a colorless crystalline solid, yield from 200 g of the chloroform extract 
solids, 12 g, 0.25% of the dried needles, [ajo^^ -27 ° (CHCI3; c 1.03); m.p. 220-222 ° C 
(lit. 200-203 °C [Balza et al. 1991^); 

FAB-MS m/z: 557 [MHf, 539 [MH-H2O]', 479 [MH-AcOHr, 435 [MH-PhCOzHr, 
417 [MH-PhCOsH-HaO]", 375 [MH-PhCOaH-AcOH]", IR (KBr) v^ax cm"': 3370, 1740, 
1650, 1600, 1585, 1450, 1370, 1265, 1180. UV 1 ^ax log s 3.01 (269 nm); log s 4.32 (223 

^H NMR (600 MHz, CHCI3, 5) Table 3-1: 0.90, s (H-19); 1.05, s (H-16); 1.30 (dd, 
J=7.2, 13.8 Hz, H-14a); 1.35, s (H-17); 1.50 (d, J=14.1 Hz, H-2a); 1.76 (s, methyl, 9- 
acetate); 1.80 (m, H-6a ); 2.0 (m, H-6p); 2.01 (s, H-18); 2.07 (s, 7-acetate methyl); 2.36 
(dd, J=14.1, 9.6 Hz, H-2p); 2.46 (dd, J=7.2, 13.8 Hz, H-14p); 2.67, br s (C-15 OH, 
exchangeable with D2O); 2.77 (br d, J=9 Hz, H-3a); 4.38 (t, J=7.2 Hz, H-13P); 4.43 (br s, 
H-5P); 4.82, s (H-20 A); 5.18, s (H-20 B); 5.57 (dd, J=4.8, 11.4 Hz, H-7a); 6.05 (poorly 
resolved br d, J=10.5 Hz, H-9a); 6.53 (d, J=10.5 Hz, H-lOp); 7.43 (t, J=7.8 Hz, H-Bz- 
meta); 7.56 (t, J=7.8 Hz, H-Bz-para); 7.87 (d, J=7.8 Hz, H-Bz-ortho). 

^^C NMR (CDCI3, 600 MHz, 5) Table 3-2: 12.0 (C-18 methyl, q); 12.9 (C-19 
methyl, q); 20.7 (7-0 acetate methyl, q) ; 21.4 (9-0 acetate methyl, q); 24.8 (C-17 
methyl, q); 26.9 (C-16 methyl, q); 29.1 (C-2, t); 36.0 (C-6, t); 37.9 (C-3, d); 45.0 (C-8, s); 
47.3 (C-14, dd); 62.4 (C-1, s); 70.3 (C-7, d); 70.9 (C-10, d); 72.4 (C-5, d); 75.9 (C-15, s); 
76.7 (C-13, d); 77.1 (C-9, d); 112.0 (C-20, t); 128.7 (C-Bz-mefa, d); 129.3 (C-Bz-/pso, s); 
129.4(C-Bz-ort/?o, d); 133.3 (C-Bz-para, d); 133.9 (C-1 2, s); 149.0 (C-4, s); 151.5 (C-11, 
s); 164.3 (CO-Ph, s); 169.9 (CO-Acetate, s); 170.5 (CO-Acetate, s). 

Analysis calculated for C31 H40O9; C, 66.89; H, 7.24. Found: C, 67.12; H, 7.35; 

Brevifoliol-5-Monoacetate [3-2] 

A mixture of brevifoliol (0.2 g); acetic anhydride (2 ml) and pyridine (0.5 ml) was 
stirred at room temperature for 2-3 min. Water was added to decompose the reagent, 
and the solid filtered after 15 min. The solid was crystallized from a mixture of acetone 
and ligroin to obtain the mono acetate as a colorless crystalline solid, yield, 0.18 g; 
m.p.224-226 °C; 

'H NMR (CDCI3, 600 MHz, 8) Table 3-1; 0.91, s (H-19); 1.02, s (H-16); 1.22 (dd, 
J=7.2, 13.8 Hz, H-14a); 1.33, s (H-17); 1.46 (d, J=14.1 Hz, H-2a); 1.76 (s, 9-0 acetate 
methyl); 1.88 m, 2.0 m (H-6); 2.06 x 2, s (methyl-18, 5-0 acetate methyl); 2.08 (s, 7-0 
acetate methyl); 2.40 (dt, J=14.1, 9.6 Hz, H-2p); 2.42 (dd, J=7.2, 13.8 Hz, H-14p); 2.75 
(d, J=9 Hz, H-3a); 2.83, br s (C-15 OH, exchangeable with D2O); 4.53 (t, J=7.2 Hz, H- 
13(3); 4.90, s (H-20 A); 5.28, s (H-20 B); 5.37 (br s, J= H-5p); 5.65 (dd, J=4.8, 11.4 Hz, 
H-7a); 6.02 (poorly resolved br d, J=10.5 Hz, H-9a); 6.63 (d, J=10.5 Hz, H-10p); 7.43 (t, 
J=7.8 Hz, H-Ph-meta); 7.56 (t, J=7.8 Hz, H-Ph-para); 7.87 (d, J=7.8 Hz, \-\-Ph-ortho). 

"C NMR (CDCI3, 600 MHz, 5) Table 3-2; 11.8 (0-18 methyl, q); 12.9 (C-19 
methyl, q); 20.8 (7-0 acetate methyl, q); 21,2 (5-0 acetate methyl, q); 21.4 (9-0 acetate 
methyl, q); 24.8 (C-17 methyl, q); 27.0 (C-16 methyl, q); 29.2 (C-2, t); 33.9 (C-6, t); 38.8 
(C-3, d); 44.8 (C-8, s); 47.1 (C-14, dd); 63.0 (C-1, s); 69.7 (C-7, d); 70.7 (G-10, d); 74.1 
(C-5, d); 75.6 (C-15, s); 76.9 (C-1 3, d); 77.9 (C-9, d); 114.0 (C-20, t); 128.7 (C-Ph-mefa, 
d); 129.2 (C-Ph-/pso, s); 129.4(C-Ph-orf/70, d); 133.3 (C-Ph-para, d); 134.0 (C-11, s); 
145.2 (C-4, s); 151.1 (C-12, s); 164.1 (CO-Ph, s); 169.9 X 2(C0-Acetate, s); 170.2 (CO- 
Acetate, s). 

Analysis calculated for C 33H 42O10; C, 66.20; H, 7.07. Found; C, 66.38; H, 7.19. 

49 [ 

Brevifoliol-5.13-Diacetate [3-4] 

The above reaction was repeated, except that it was heated at 80-90° C (water 
bath) for 30 min. After cooling, water was added and the solid filtered after 10 min. The 
solid was crystallized from acetone/ ligroin to give the diacetate as a colorless crystalline 
solid, yield, 0.2 g; m.p.241-243°C; 

^H NMR (CDCI3, 600 MHz, 5) Table 3-1; 0.92, s (H-19); 1.11, s (H-16); 1.25 (dd, 
J=7.2, 13.8 Hz, H-14a); 1.35, s (H-17); 1.46 (d, J=14.1 Hz, H-2a); 1.75 (s, 9-0 acetate 
methyl); 1.90 (m, H-6a) 2.0 (m, H-6p); 2.02 (s, 5-0 acetate methyl); 2.03 (s, 13-0 
acetate methyl); 2.07 (s, 18 methyl); 2.08 (s, 7-0 acetate methyl); 2.41 (dd, J=14.1, 9.6 
Hz, H-2p); 2.51 (dd, J=7.2, 13.8 Hz, H-14(3); 2.72 (d, J=9 Hz, H-3a); 2.74, br s (C-15 OH, 
exchangeable with D2O); 4.92, s (H-20 A); 5.28, s (H-20 B); 5.39 (br s, J= H-5p); 5.54 (t, 
J=7.2Hz, H-13P); 5.61 (dd, J=4.8, 11.4 Hz, H-7a); 6.09 (poorly resolved br d, J=10.5 Hz, 
H-9a); 6.65 (d, J=10.5 Hz, H-10p); 7.43 (t, J=7.8 Hz, H-Ph-mefa); 7.56 (t, J=7.8 Hz, H- 
Ph-para); 7.87 (d, J=7.8 Hz, H-Ph-ortho). 

'^C NMR (CDCI3, 600 MHz, 5) Table 3-2: 11.9 (C-18 methyl, q); 12.9 (C-19 
methyl, q); 20.7 (7-0 acetate methyl, q); 21.0 (13-0 acetate methyl, q); 21.2 (5-0 
acetate methyl, q); 21.4 (9-0 acetate methyl, q); 24.8 (C-17 methyl, q); 27.0 (C-16 
methyl, q); 29.1 (C-2, t); 33.9 (C-6, t); 38.8 (C-3, d); 44.8 (C-8, s); 44.1 (C-14, dd); 63.0 
(C-1, s); 69.6 (C-7, d); 69.8 (C-10, d); 74.1 (C-5, d); 75.6 (C-15, s); 76.9 (C-13, d); 79.3 
(C-9, d); 114.3 (C-20, t); 128.8 {C-Ph-meta, d); 129.1 (C-Ph-/pso, s); ^29.5{C-Ph-ortho, 
d); 133.4 (C-Ph-para, d); 136.4 (C-11, s); 145.2 (C-4, s); 147.3 (C-12, s); 164.1 (CO-Ph, 
s); 169.6 (CO-Acetate, s); 169.9 (CO-Acetate, s); 169.91 (CO-Acetate, s); 170.5 (CO- 
Acetate, s). 

Analysis calculated for C 35H 44O11: C, 65.61 ; H, 6.92. Found: C, 65.68; H, 6.99. 


Brevifoliol-13-Ketone [3-61 

A solution of brevifoliol (0.2 g) in benzene was treated with Mn02 (manganese 
dioxide, 1 g, Fisher Scientific) and the mixture heated under reflux for 2 hours, at which 
time, the starting material was consumed and a slightly faster moving product was 
formed. The mixture was filtered, concentrated and applied to a small silica column (15 
g) in dichloromethane. Elution with 1% acetone in dichloromethane gave the major 
product which was recovered by concentration as a colorless powder, yield, 0.1 2g. The 
^H- and ^^C NMR spectra of this faster moving product were quite poorly resolved and ; 

only gave usable results at temperatures below -10 ° C. Recrystallization and further 
chromatography failed to improve this situation, and low temperature NMR experiments 
indicated that a rotameric equilibrium was responsible for the poor resolution seen in 
these spectra. 

^H NMR (CDCI3, 600 MHz, -40 °C, 5) major rotamer: 0.92, s (H-19); 0.98, s (H- 
16); 1.35, s (H-17); 1.46 (d, J=14.1 Hz, H-2a); 1.78 (s, 9-0 acetate methyl); 1.90 (m, H- 
6a) 2.0 (m, H-6p); 2.01 (s, 18 methyl); 2.02 (s, 7-0 acetate methyl); 2.32 (d, J=19.0, 
14a); 2.48 (d, J=19.0 Hz, H-14p); 2.92 (unresolved, H-3a); 2.74, br s (C-15 OH, 
exchangeable with D2O); 4.92, s (H-20 A); 5.28, s (H-20 B); 5.39 (br s, J= H-5p); 5.54 (t, 
J=7.2Hz, H-13P); 5.61 (dd, J=4.8, 11.4 Hz, H-7a); 6.09 (poorly resolved br d, J=10.5 Hz, 
H-9a); 6.65 (d, J=10.5 Hz, H-10p); 7.43 (t, J=7.8 Hz, H-Ph-meta); 7.56 (t, J=7.8 Hz, H- 
Ph-para); 7.87 (d, J=7.8 Hz, H-Ph-ortho). 

^^C NMR (CDCI3, 600 MHz, 5): 8.9 (C-18 methyl, q); 12.5 (C-19 methyl, q); 20.7 
(7-0 acetate methyl, q); 21.0 (9-0 acetate methyl, q); 26.2 (C-17 methyl, q); 26.6 (C-16 
methyl, q); 27.3 (C-2, t); 27.7 (C-6, t); 34.2 (C-3, d); 43.2 (C-8, s); 48.3 (C-14, t); 58.1 (C- 
1, s); 70.8 (C-7, d); 71.0 (C-10, d); 73.2 (C-9, d); 75.6 (C-15, s); 111.4 (C-20, t); 128.8 
(Ph-meta, d); 129.1 (Ph-ipso, s); 129.5(Ph-ortho, d); 133.4 (Ph-para, d); 136.4 (C-11, s); 


145.2 (C-4, s); 144.3 (C-12, s); 163.1 (C-11, s); 165.4 (CO-Ph, s); 169.3 (CO-Acetate, s); 

170.3 (CO-Acetate, s); 207.5 (C-13 Ketone, s). 

Analysis calculated for C 31 HssOg: C, 67.13; H,6.91. Found: C, 67.48; H, 6.97. 
Dihydrobrevifoiio! [3-7] 

A solution of brevifoliol (0.2 g) in ethyl acetate (10 ml) was hydrogenated in a 
Parr apparatus using Platinum oxide (0.05 g) for 16 liours. TLC revealed the formation 
of a slightly slower moving product. The mixture was filtered and the filtrate 
concentrated to dryness and purified by chromatography on silica gel in 
dichloromethane. Elution with 2% acetone in dichloromethane gave a minor product, 
which was not further investigated. The fractions eluted with 2-5% methanol in 
dichloromethane gave the major product, which was obtained as a colorless powder, 
yield, 0.1 g. 

Reduction of the 4(20) double bond resulted in significant broadening of most 
peaks in the ^H- and ^^C NMR spectra, but the appearance of additional signals from 
methyl group at C-20 and methylene at C-4 could be seen, as well as the loss of the two 
characteristic exocyclic methylene singlets. 

Analysis calculated for C31H42O9: C, 66.60; H, 7.60. Found: C, 66.89; H, 7.88. 
Brevifoliol Epoxide [3-8] 

A mixture of brevifoliol (0.3 g) and meta-chloroperoxybenzoic acid (MCPBA, 0.2 
g) in toluene (15 ml) was heated under reflux for 30 min. After cooling, the mixture was 
diluted with ether, washed successively with aqueous sodium bisulfite, aqueous sodium 
bicarbonate and saline, and the organic layer concentrated to dryness. The solid was 
crystallized from acetone/ligroin, to give a colorless crystalline epoxide, yield, 0.15 g; 
m.p. 227-230 °C. 


'H NMR (CDCI3, 600 MHz, -40° C, 5) : 1.01 (H-19, s); 1.09 (H-16, s); 1.24 (dd, 
J=7.2, 13.8 Hz, H-14a); 1.27 (H-17, s); 1.40 (H-2a, br d J=14.1 Hz); 1.76 (s, methyl, 9- 
acetate); 1.80 (m, H-6a ); 2.0 (m, H-6p); 2.01 (s, H-18); 2.07 (s, 7-acetate methyl); 2.36 
(dd, J=14.1, 9.6 Hz, H-2p); 2.46 (dd, J=7.2, 13.8 Hz, H-14p); 2.67, br s (C-15 OH, 
exchangeable with D2O); 2.64 (H-3a, br d, J=9 Hz); 2.72 (C-20, s); 3.59 (C-20, s); 4.20 
(br s, H-5p); 4.46 (H-13p, br d, J=7.2 Hz); 5.57 (H-7a, br d, J=4.8, 11.4 Hz); 6.05 (H- 
9a, poorly resolved br d, J=10.5 Hz); 6.54 (H-lOp, br d, J=10.5 Hz); 7.43 (Ph-meta, t, 
J=7.8 Hz); 7.56 (Ph-para, t, J=7.8 Hz); 7.87 (Ph-ortho, d, J=7.8 Hz). 

"C NMR (CDCI3, 600 MHz, -40° C, 5) : 11.9 (C-18 methyl, q); 12.9 (C-19 methyl, 
q); 20.7 (7-0 acetate methyl, q) ; 21.3 (9-0 acetate methyl, q); 23.9 (C-2, t); 25.0 (C-17 
methyl, q); 27.1 (C-16 methyl, q); 34.1 (C-3, d); 34.4 (C-6, t); 45.4 (C-8, s); 46.4 (C-14, t); 
50.1 (C-4, s); 60.2 (C-20, t); 62.4 (C-1, s); 69.5 (C-7, d); 70.5 (C-10, d); 71.7 (C-5, d); 
75.8 (C-15, s); 76.7 (C-13, d); 77.1 (C-9, d); 128.7 (Ph-meta, d); 129.4 (Ph-ipso, s); 
129.5(Ph-ortho, d); 133.2 (Ph-para, d); 134.3 (C-12, s); 151.3 (C-11, s); 164.3 (CO-Ph, 
s); 169.96 (CO-Acetate, s); 170.0 (CO-Acetate, s). 

Analysis calculated for C31H40O10: C, 65.02; H, 7.04. Found: C, 64.72; H, 7.24. 
Ozonization of Brevifoliol: Brevifoliol-norketone [3-9] 

A solution of brevifoliol (1 g) in a 9:1 mixture of chloroform and methanol (25 ml) 
was cooled in a dry ice/ acetone bath and saturated with ozone produced by an ozonizer 
(Ozone Research and Equipment Co., Phoenix, AZ). After testing for the absence of the 
starting material by TLC, the mixture was removed from the bath and treated with 
dimethyl sulfide (1 ml) and let stand at room temperature for 2 h. It was then 
concentrated to dryness and applied to a silica column prepared in chloroform. Elution 
with 2% acetone in chloroform gave two bands, which were separated and the fractions 
concentrated separately. 


The faster moving fraction [3-9] was obtained as a colorless, amorphous powder, 
yield, 0.25 g. 

^H NMR (CDCI3, 600 MHz, -40 ° C, 5) major rotamer: 1.02, s (H-19); 1.05, s (H- 
16); 1.35, s (H-17); 1.46 (d, J=14.1 Hz, H-2a); 1.78 (s, 9-0 acetate methyl); 1.98 (m, H- 
6a); 2.0 (m, H-6p); 2.01 (s, 18 methyl); 2.02 (s, 7-0 acetate methyl); 2.32 (d, J=19.0, 
14a); 2.48 (H-14p, dd, J=14, 7.5 Hz); 3.19 (H-3a, d, J=10.2); 4.20 (H-5p, br s); 4.48 (H- 
13p, br t, J=7.2 Hz); 5.78 (H-7a, br m); 5.92 (H-9a, br); 6.52 (H-10p, br d, J=10.5 Hz); 
7.43 (Ph-meta, t, J=7.8 Hz); 7.56 (Ph-para, t, J=7.8 Hz);7.87 (Ph-ortho, d, J=7.8 Hz). 

^^C NMR (CDCI3, 600 MHz, 5): 12.1 (C-18 methyl); 14.2 (C-19 methyl); 20.6 (7- 
O acetate methyl); 21.2 (5-0 acetate methyl); 21.3 (9-0 acetate methyl); 24.8 (C-17 
methyl); 27.0 (C-16 methyl); 29.1 (C-2); 33.9 (C-6); 34.8 (C-3); 46.4 (C-8); 46.4 (C-14); 
62.0 (C-1); 69.9 (C-7); 70.7 (C-10); 72.0 (C-5); 75.9 (C-15); 76.9 (C-13); 77.3 (C-9); 
128.8 (Ph-meta); 129.1 (Ph-ipso); 129.5(Ph-ortho); 133.4 (Ph-para); 134.4 (C-11); 144.5 
(C-4); 147.3 (C-12); 164.3 (CO-Ph, s); 169.8 (CO-Acetate, s); 170.0 (CO-Acetate, 

Analysis calculated for C30H38O10: C, 64.50; H, 6.86. Found: C, 64.68; H, 6.97. 

The slower moving fraction was obtained as a colorless crystalline solid, yield, 
0.3 g; m. p. 225-232 ° C. It was found to be identical with the epoxide [3-8], described 
Brevifoliol-4,20-Diol [3-101 

To a solution of brevifoliol (0.4 g) in pyridine (10 ml) was added osmium tetroxide 
(0.2 g) and the reaction mixture stirred for 1 h, after which time, the starting material was 
replaced by a much slower moving component. After decomposing the excess reagent 
with a solution of sodium bisulfite in pyridine, water and dilute sulfuric acid were added 
and the mixture extracted with dichloromethane. After concentration, the product was 


placed on a silica column in dichloromethane. Elution with 2% methanol in 
dichloromethane gave the major band which yielded [3-8] as a white powder, final yield, 
0.12 g. 

Analysis calculated for C31H42O11: C, 63.04; H, 7.17. Found: C, 62.88; H, 7.25. 
Saponification of Brevifoliol [3-11] 

A solution of brevifoliol (1 g) in methanol (20 ml) was stirred with IN potassium 
hydroxide (10 ml) for 1 h. TLC showed that the starting material was absent and very 
slow moving, non UV-absorbing component produced. The reaction mixture was 
passed through a small column or Amberlite-IR120 ( a sulfonic acid resin) in the H+ 
form. The column was washed with 1:1 methanol/water. The effluent and washes were 
concentrated to dryness and the solid crystallized from acetone to give [3-11] as a 
colorless crystalline solid, yield, 0.45 g; m.p. 290 °C dec. 

Analysis calculated for C20H32O6+H2O: C, 62.15; H, 8.87. Found: C, 62.48; H, 
Debenzoyl Brevifoliol-Pentaacetate [3-12] 

Compound [3-11] (0.2 g) was acetylated using acetic anhydride (2 ml) and 
pyridine (0.5 ml) by heating at 80 ° C for 30 min. Water was added to decompose the 
reagent and the solid filtered. It was crystallized from ether/ ligroin, yield, 0.2 g; m.p. 
184-1 87 °C. 

'H NMR (CDCI3, 5): 6.36 (H-10, d, J=10.2); 5.88 (H-9, br d, 10.2); 5.54 (H-13p, t, 
J=7.2); 5.53 (H-7a, q, J=5.4, 10.2); 5.36 (H-5|3, br s); 5.26 (H-20b, s); 4.87 (H-20a, s); 
2.65 (OH-15, s); 2.64 (H-3a, d, J=9); 2.48 (H-14p, dd, J=13.8, 7.2); 2.36 (H-2a, dd, 
J=14.1, 9.3); 2.07 (AcMe, s); 2.06 (AcMe, s); 2.02 (AcMe, s); 2.00 (AcMe, s); 1.97 
(AcMe, s); 1.95 (18-Me, s); 2.0 (H-6a, m); 1.85 (H-6[3, cm); 1.42 (H-2|3, d, J=14.4); 1.31 
(H-17 Me);1.22 (H-14a, dd, J=13.8, 7.2); 1.13 (H-16 Me); 0.88 (H-19 Me). 


IR V max (KBr, cm"^): 3565 (OH, sharp); 2960, 1740 (C=0); 1370, 1230, 1030. 
MS(FAB): 580 [MH+]. 
Periodate Oxidation of [3-1 11 to [3-13] 

A solution of [3-11] (0.2 g) in methanol (5 ml) was treated with sodium periodate 
(0.3 g) in IN sulfuric acid (2 ml). After 30 min, TLC showed that the starting material 
was absent and was replaced by a faster moving product visible under the UV light, 
unlike the starting material. After dilution with water, the mixture was extracted with ethyl 
acetate and the extract concentrated to dryness. The crude dialdehyde [5-13] was not 
further purified before the next reaction, but did exhibit signals for two aldehydes the ^H 
spectra. The only significant changes from the parent compound showed the loss of the 
isolated ^H spin system from the protons on C-9 and C-10, and the conversion of two 
hydroxyl carbons into aldehydes (Guthrie, 1961). 

'H NMR (CDCIs.S) : 9.96 (CHO, s); 9.42 (CHO, s); 5.36 (H-20b, s); 5.02 (H-20a, 
s); 4.58 (H-5a, m); 4.40 (H-13a, br t); 2.60 (H-3(3, d); 1.30 (Me, s); 1.25 (Me, s); 1.02 
(Me, s); 0.88 (Me, s). 

Analysis calculated for C30H42O11: C, 62.27; H, 7.32. Found: C, 62.10; H, 7.44. 
Formation of Osazone [3-141 from [3-131 with 2.4-DNPH 

Dialdehyde [3-13] was dissolved in methanol, (2 ml) and heated with a solution of 
2,4-dinitrophenyl hydrazine (0.1 g) in 2N hydrochloric acid (2ml) and methanol (2 ml). 
After 2 h at room temperature, the mixture was extracted with chloroform and the 
concentrated extract chromatographed on a silica column. The major band [3-14] was 
obtained as an orange yellow crystalline solid, m.p. 215-218 °C. Both ^H- and "C NMR 
spectra indicated the retention of all twenty of the carbons from the atieo-taxane 


Debenzovl Brevifoliol [3-15] 

A solution of brevifoliol (0.3 g) in 30% methanol in water (10 ml) was heated in a 
sealed tube at 135 ° C for 90 min. The cooled mixture was neutralized with sodium 
bicarbonate and extracted with chloroform. The extract was purified by chromatography 
on a C-8 reverse phase column using a step gradient of 25-60% acetonitrile in water in 
5% increments of solvent concentration. Elution with 30-35% acetonitrile in water gave 
the major product, which was obtained as a white powder, yield, 0.1 g. The ^H and ''^C 
NMR spectra revealed the loss of the benzoate from the C-10 position and retention of 
the acetate substituents at C-9 and C-7 

Analysis calculated for C24H36O8: C, 63.70; H, 8.02. Found: C, 63.89; H, 7.88. 

Benzoic acid was also isolated from the reaction mixture and confirmed using 
NMR and UV analyses. 


The isolation and structural elucidation of brevifoliol [3-1] was described in detail 
in Chapter 3. Brevifoliol occurs to the extent of 0.2-0.3 % in the needles of T. brevifolia, 
and to a lesser extent in the bark of the same species, in the needles of T. x media 
Hicksii (Rehd.) and in the needles of T. walllchlana. (Georg e^ al. 1993). Large 
quantities of the crystalline compound can be readily isolated from the needles of T. 
brevifolia, which is the best source for the compound. 

However, other than acetylation to a diacetate (Balza e^ al. 1991); and the 
attachment of the N-benzoyI phenyl-isoserine side chain at the C-13 position (Georg, et 
al. 1993); no record of its reactions reflecting its vahous functional groups has been 
published. This paucity of such information is in keeping with the current trend that, in 
spite of the virtual explosion of new taxanes that were isolated and characterized 
structurally by spectral data over the past five years. Very few have been investigated 
for their chemical reactions, for the relative reactivities of similar functional groups, or for 
any unusual reactions as a consequence of their stereochemical disposition. 

Understanding the products of the various transformations that a compound can 
be subjected to and the relative rates of reaction can lead to important insight 
concerning entity, and for the general advancement of chemistry. Although the structure 
of brevifoliol is now well established as a result of spectral and x-ray crystallographic 
data, we can gain insight into this molecule through reactions such as those described in 
Chapter 3. In addition to these, a number of reactions also carried out for the purpose of 



structural elucidation of [3-1] took unexpected courses. The products of these reactions 
were isolated and characterized structurally, and the details are described here. 
1 . Acid-Catalyzed Acetylation 

In Chapter 3, acetylation of [3-1] by means of acetic anhydride and pyridine to 
give the crystalline monoacetate [3-2] and the diacetate [3-4] has been described. We 
isolated fronn the needles of T. brevifolia another taxane, which resembled brevifoliol, 
giving a dark greenish blue color when sprayed with sulfuric acid and heated. This same 
product was also subsequently isolated by others (Barboni et al. 1993). It was similar to ! 

but different from the monoacetate [3-2], but when acetylated, gave [3-4], thus showing | 


that it was the 1 3-acetate [3-3]. j 



Acetylation of brevifoliol using acetic anhydride and BF3 gave a different | 

crystalline product, with almost the same Rf as that of the diacetate [3-4]. The BF3 
product closely resembled the 5, 13-diacetate in its NMR spectral properties also, but 
with some differences. In the ^H NMR spectrum, the singlet at 52.74 which is assigned 
to the 15-hydroxyl in the diacetate [3-4], was no longer present. The two methyl singlets 
assigned to C-1 6 and C-17 at 51.11 and 51.35 were deshielded to 61.63 and 51.71. The 
other two (C-1 8 and C-1 9) showed either no shift or a much smaller one (5 2.03->5 1.96 
for C-1 8). Thus, the significant downfield shift of the signals due to C-1 6 and C-17 
suggested that acetylation of the 15-hydroxyl might have taken place. 

Additionally, the signals due to the H-2a and 2p showed slight downfield shifts 
(H-2a: 51.46, broad doublet, J=13 Hz to 1.53 ppm; H-2p: 52.41, doublet of doublets, 
J=9,13 Hz to 2.65 ppm). The spectra of brevifoliol and its acetates generally show the 
H-10a signal as a sharp and well resolved doublet, but the one due to H-9p as a broad, 
poorly resolved doublet. In the BFs-reaction product, the signal due to H-9p was not only 
a well resolved doublet (J=10.8 Hz); but also shielded by 0.29 ppm to 5 5.80. These 


observations suggest that the C-15 hydroxy! might be responsible for the blurring of the 
signal of H-9p, and acetylation of this hydroxyl has eliminated that interaction. 

Further support for the acetylation having taken place at the C-15 hydroxyl is 
shown by the appearance of another acetyl methyl signal at 5 2.11 in the ^H NMR 
spectrum. The appearance of two more signals in the "C NMR spectrum was also 
evident, in which 5 acetyl methyl and 5 acetyl-carbonyl signals were present, with the 
fifth one at 5 21.7 and 5 169.5, respectively. Also in the ^^C NMR spectrum, a significant 
downfield shift from 5 75.6 to 5 87.2 for the C-15, and an upfield shift from 5 27.0 to 5 
24.8 and from 5 23.1 to 5 22.0 for the signals due to C-16 and C-17 respectively, 
completes the evidence to indicate that the 15-hydroxyl was acetylated to give [3-5]. 

A comparison of the ^^C NMR spectral data of brevifoliol, the 5-monoacetate 
[3-2], the 13-monoacetate [3-3] (naturally occurring and confirmed through semi- 
synthesis); the 5, 13-diacetate [3-4] and of the BFa-catalyzed acetylation product [3-5] 
were shown in Chapter 3 in Table 3-1. 
2. Oxidation 

Oxidation of brevifoliol with manganese dioxide gave the monoketone, the NMR 
spectral data of which showed that the 13-hydroxyl was oxidized, leading to the structure 
[3-6], as described in Chapter 3. Oxidation of [3-1] with Jone's reagent gave initially, the 
same 13-monoketone [3-6], but on further reaction, this was replaced by a faster moving 
compound [4-1], whose spectral data pointed to an unexpected course of reaction. 

The molecular formula of the product, C31H36O9 (MH+, 553) indicated the loss of 
4 protons, as compared to brevifoliol. Although this might indicate that both hydroxyls 
were oxidized to give the diketone [4-2], certain features suggested othen/vise. To begin 
with, the ^H NMR spectrum showed broad peaks which indicated the existence of a 


rotameric equilibrium, which was confirmed by a spectrum tai<en at -40 ° C, in which two 
sets of peaks with a 5:1 ratio were seen. 

In the major rotamer, the coupling between the C-9 and C-10 protons was found 
to be 4 Hz, in contrast to the value of 10.5 Hz shown by brevifoliol [3-1], (a similar 
diketone prepared from 2-acetoxy brevifoliol (taxchinin A); described by Fuji et al. (1992) 
and Appendino et al. (1993) also showed a coupling of 4 Hz. Next, a singlet appeared at 
9.4 ppm, which interacted in the HETCOR spectrum with the peak at 194 ppm. The 
latter showed a negative signal in the Attached Proton Test (APT). These observations 
indicated the presence of an aldehyde functionality, presumably at C-20. Additionally, in 
the spectrum of the diketone such as [4-2], the C-5 proton signal was absent, as 
expected, and the exocyclic methylene protons appeared as two singlets at 5 5.06 and 5 
5.94 ppm. The corresponding carbon signals appearing at 5127 and 5143.4 ppm. 

However, in the Jones oxidation product, the singlets due to the exocyclic 
methylene protons were absent, and the charactenstic C-20 carbon signal which 
appears in the 6110-5120 ppm region was also missing. Instead, a signal was found at 
5 6.73 ppm, which interacted with the signals at 62.5 and 52.7 (C-6 protons); and in the 
HETCOR spectrum, with the signal present at 6 147 ppm, and this latter gave a negative 
signal in the APT spectrum. These data seem to suggest that the product is not the 
5,13-diketone [4-2], but an aldehydic product with a double bond present at C-4/C-5, as 
shown in [4-1]. 

One possible explanation is that the sulfuhc acid in the reagent caused hydration 
of the 4/20 double bond, and the primary alcohol so generated was oxidized to the 
aldehyde, followed by dehydration to yield the 4/5-double bond. 



n O „A> 

[4-1 ] - Jones Oxidation - Aldehyde 

[4-2] - Mn02 Diketone 

Figure 4-1 : Oxidation Products 

3. Action of BF ^ on Brevifoiiol [4-31 

Before the structure of brevifoiiol was fully established, the possibility that the two 
hydroxyls present in the compound might be vicinal to each other was considered. With 
the aim of forming an isopropylidene derivative, brevifoiiol was reacted with acetone in 
the presence of Dowex-50 (sulfonic acid resin) as the catalyst. Reaction took place 
readily with the formation of a faster moving product, with some decomposition also 
taking place (colors). The reaction proceeded with less decomposition when BF3- 
etherate was used as the catalyst. 

Chromatography of the mixture from either reaction yielded the major product as 
a colorless crystalline solid. Its NMR spectrum quickly ruled out the possibility of its 
being an isopropylidene derivative. The FAB-mass spectrum gave a value for the MH+ 
m/z of 481, as compared with 557 for brevifoiiol, thus showing a loss of 76 mass units. 
The elemental analysis, which agreed with C28H32O7 showed a loss of C3H8O2 compared 
to brevifoiiol. This may be interpreted as the loss of the C3H7O- side chain attached to 
C-1 , as well as that of H2O, possibly through the elimination of the C-1 3-OH (or C-5-0H). 
The evidence to support this assertion is given below. 


1 . The most striking evidence is that only two of the four methyl signals that are seen in 
brevifoliol appear in the BFa-reaction product. This is not only observed in the ^H 
spectrum, but also in the ^^C NMR thus showing that the oxy-isopropyl chain at C-1 
is not present. 

2. An examination of the isolated spin system: 5p - 6a- 6p - 7a in the ^H NMR spectrum 
of brevifoliol and that of the product of BF3 reaction indicated that the free hydroxyl at 
C-5a was still present, but that the C-1 3a-0H was absent. Through a study of the 
interactions in the COSY spectrum, it was possible to assign (and distinguish) the 
signals for C-6 and C-14 in the region below 53. 

3. A new methine proton signal appeared as a broad singlet at 55.84, assigned to H-13, 
which interacted in the HETCOR spectrum with the signal at 5124.1, which 
corresponds to C-1 3. 

4. The DEPT spectrum showed the presence of four signals for methyl-type (CH3) 
carbons (51 1.4,51 3.5 for C-1 9 and C-1 8, 5 20.7, 5 21 .2 for the two CO-Me); two 
signals for methylene-type (CH2) carbons (5 1 12.4 for C-20, 5 44.6 for C-14, 5 34.1 
for C-6 and 5 27.9 for C-2, nine signals for methine-type (R3CH) carbons (5133.0, 
51 29.7, 51 28.4 for the five aromatic carbons, four aliphatic CH-OR type carbons 
[573.2 (C-9); 572.5 (C-5); 579.6 (C-7); 667.4 (C-10)], one aliphatic methine type 
carbon (539.6 (C-3)); one unsaturated methine type carbon (5124.1 (C-1 3) and nine 
quaternary carbons (three carbonyls, one aromatic C carrying the carboxyl, C-1(?); 
C-4, C-11, C-1 2 and C-8); together, account for the 28 carbons present.. 

5. HETCOR interactions supported the assignments for the o, m, p- positions in the 
benzoate, and for the C-1 3, C-20, C-9, C-5, C-7, C-10. C-3, C-6, C-2, C-1 8, C-1 9 
and the two acetate methyls. 


6. In the ID nOe-difference spectrum, the interaction between the C-14a,p and C-13 p 
protons was the strongest. Crowding of the region around 82.8 ppm made the 
spectrum more difficult to interpret, and not very informative. 

Table 4-1 : NMR Spectra of Connpound [4-3] from BF3 Reaction 












1.96 m 





3.00 m 




2.70 m 









4.42 br s 





1.76 m 





2.05 m 




5.48 brd 










5.34 d 6.0 





6.30 d 6.0 














5.83 brs 




14 a, p 

2.85-3.0 cm 





2.06 s 





1.23 s 




20 A 

4.98 brs 




20 B 

5.20 brs 














8.01, d 7.5 





7.44, t 7.5 





7.56, t 7.5 





1.98 s 
2.00 s 







'H NMR were recorded at 600 MHz and "C NMR at 150 MHz in CDCI3 on 
a Varian Unity 600 spectrometer at ambient temperature. Chemical shifts 5 (ppm) 
are reported relative to TMS as an internal standard. 



[4-3] - BFg-etherate Product 
Figure 4-2 : BFs-etherate Catalyzed Elimination Product 

Based on this reasoning, the structure of the BFs-reaction product was assigned 

as shown [4-3] in Figure 4-2 above. The DEPT spectra of [4-3] are given in Figure 4-3. 

CHS Carbons 



CH2 Carbons 


14 6 2 

J L 

CH Carbons 



5 7 




AdI Protonated Carbons 

V II ' ■ ' Itjl 

11 I 1 r 1 1 r-] 1 in i i ; i i | i : ■ ; r r ri \- ] v \ i i ; r i i :■ ; i i ; : | r i i i ] : i I i ' i ; v r [ t ri i -i r i t i ;■ i "v i i ' 
140 120 ICO 80 SO -lb P.'o d 

Figure 4-3 : DEPT Spectra of BF3 Elimination Product [4-3] 


A reaction such as this has not been reported in the taxane series, resulting in 
the loss of the oxy-isopropyl side chain. In taxol and related compounds containing the 
conventional taxane skeleton, action of Lewis acids such as BF3 was studied and is 
shown to produce one or two different changes, depending on the whether protic or 
aprotic solvent is used. In one case, isomerization of the A-ring from a 6- to a 5- 
membered A ring takes place with the oxy-isopropyl group attached at C-1. In the 
second instance, the oxetane ring is opened to form a diol, or with the acetate group 
migrating from C-4 to C-20 to give the 4-hydroxy-20-acetoxy compound. The reaction 
described here appears to be a continuation of the action of the Lewis acid on the 5- 
membered A ring, with the elimination of the oxy-isopropyl substituent. 
4. Reaction with Iodine/Silver Acetate [4-4] 

Once the structure of brevifoliol had been elucidated, the value and usefulness of 
this relatively abundant compound in the needles of T. brevifolia was considered. Since 
the addition of the N-benzoyI isoserine side chain at C-1 3 did not generate activity in the 
final product, it was reasoned that the oxetane ring at the 4/20 position might be 
necessary to produce activity. To this end, one approach was investigated, involving the 
use of iodine in some form to add across the 4/20 double bond and thereby permitting 
substitution with other groups. 

Brevifoliol was found to react readily with bromine, but the reaction yielded 
multiple products and considerable decomposition. Reaction with iodine was similarly 
complex and led to much decomposition and dark colored products. With the idea that 
addition of a silver salt which can remove the acid that might be produced, but not be too 
strongly basic (e.g. silver oxide) and hence hydrolyze the ester functions, silver acetate 
was selected for use with the iodine. The remote possibility that silver acetate might 


displace the iodine located at C-4 to produce the 4-acetoxy-20-iodo compound was also 
attractive (Woodard & Brutcher, 1958). 

When brevifoliol was stirred with iodine and silver acetate, the course of the 
reaction was clearly different. No multiple products or decomposition to dark colored 
products was seen, even when the reaction was continued for 15-30 hours, unlike the 
reaction with iodine alone that became dark in 1-2 hours. The reaction was continued 
until the starting material was consumed and a major, faster moving compound was 
produced. Chromatography on a silica column gave a colorless crystalline solid. 

Acetylation with acetic anhydride and pyridine at 70° C for 15 minutes produced 
the 5-0-monoacetate [4-5], confirming the presence of the 5 hydroxyl with NMR 
analysis. Treatment of [4-4] with n-Bromosuccinimide produced the 5-0 ketone, further 
confirming the absence of a free C-13 hydroxyl on the basis of NMR and UV spectral 

[4-4]- Iodine/Silver Acetate R-oduct 

[4-5] - Acetate 

Figure 4-4 : Iodine/Silver Acetate Product [4-4] and Acetate 







10 9 



^ 14/14 






.. i Li 


20 2 

-1 1 r 



1 ^ ^ ^ 1 ^ 

2 PPM 

Figure 4-5 : H,H-COSY Spectrum of [4-4] 

The ^H NMR spectrum was similar to brevifoliol in most respects, with a few 
revealing differences. The AB quartet normally seen from H-2p became a clean doublet 
(J=13.8 Hz) deshielded from 5 2.36 to 5 3.31, and coupled to the H-2a proton, which was 
deshielded from 5 1.49 to 5 2.34. This deshielding and the coupling patterns indicated 
the loss of H-3P with the possible formation of a double bond between C-3 and C-4. 





Figure 4-6 : HETCOR Spectrum of [4-4] 

Recorded on Varian VXR 300 spectrometer at 75.432 MHz inCDCIa with TlViS as 
internal standard. Fi: 3000.3 Hz; F2: 15528 Hz; Acquisition time 65.9 sec; Di; 1 sec; 
Ambient temperature; Decouple proton; Level 70 higli power; PW: 90°; 128 repetitions X 
128 increments; Waltz 16 modulation; pseudo ecino; FT size: 2K X 512 data points; time: 
5 hours. 

Elemental analysis and FAB-MS gave the molecular formula CaiHseOg, which 
indicated essentially that loss of one molecule of water and dehydrogenation had taken 


place. The presence of a hydroxyl was indicated by the fact that the compound would 
still undergo acetylation to form a monoacetate. 

The C-15 hydroxyl was still present in both the carbon and proton spectra, and 
the only other significant change in the spectra occurred with the C-20 signals. The two 
broad singlets normally seen around 5 ppm were replaced by an isolated spin system of 
doublets at 4.35 and 3.92 which resemble the H-20 oxetane pattern seen in taxol. 


Brevifoliol Triacetate [3-5] 

To a solution of brevifoliol (0.2 g) in acetic anhydride (2 ml) was added 0.2 ml of 
a 2% solution of boron trifluoride etherate in acetic anhydride to give a final 
concentration of 0.2% of boron trifluoride etherate. After 20 min at room temperature, 
the mixture was diluted with water. After another 10 min. the solid was separated, 
washed, taken up in ether and concentrated to dryness. The solid was crystallized from 
ether in ligroin to give [3-5] as a colorless crystalline solid, yield, 0.2 g; m.p. 214-216 ° C. 
Oxidation with Jones Reagent to [4-1] 

A solution of brevifoliol (0.2 g) in acetone (10 ml) was treated with Jones reagent 
(2 ml) added in small portions and with stirring. Initially TLC analysis of the reaction 
mixture showed that a yellow color giving spot appeared above that of the starting 
material. Gradually the first product changed into an even faster moving component. 
When this latter was the predominant product, the reaction was stopped by the addition 
of water and extraction with chloroform. After concentration of the solvent, the product 
was chromatographed on a silica column in 1:1 chloroform/ ligroin. Elution with 
chloroform gave the major component, which was obtained as a colorless crystalline 
solid, yield, 0.05 g; m.p. 234-236 ° C. 



Action of Boron Trifluoride on Brevifoliol [4-31 

Brevifoliol (0.3 g) was dissolved in acetone (10 ml) and to the solution was added 
1 ml of 1% boron trifluoride etherate in acetone to make a 0.1% overall concentration of 
boron trifluoride in the reaction mixture. After 3 h, water was added, the solid filtered 
and after drying, subjected to chromatography on silica gel in chloroform/ ligroin (1:1). 
The major band obtained with the same solvent was crystallized from ether/ligroin, yield, 
0.1 g, m.p. 162-165 °C. 

^H NMR (CDCI3, Varian Unity 600 MHz, 5): 1.16, s (H-19); 1.03, s (H-16); 2.08 
(cm, H-14a); 1.27, s (H-17); 2.32 (d, J=13.8 Hz, H-2a); 1.92 (s, methyl, 9-acetate); 1.84 
(m, H-6a ); 1.98 (m, H-6p); 2.26 (s, H-18); 2.05 (s, 7-acetate methyl); 3.30 (d, J=13.8 Hz, 
H-2p); 2.28 (cm, H-14P); 2.95, br s (C-15 OH, exchangeable with D2O); 4.38 (t, J=7.2 
Hz, H-13P); 4.28 (br s, H-5p); 4.54 (d, J=13.2 Hz, H-20 B); 4.54 (d. J=13.2 Hz, H-20 B); 
5.51 (dd, J=4.8, 11.4 Hz, H-7a); 6.07 (d, J=10.1 Hz, H-9a); 6.54 (d, J=10.1 Hz, H-lOp); 
7.45 (t, J=7.5 Hz, H-Bz-mefa); 7.57 (t, J=7.5 Hz, H-Bz-para); 7.89 (d, J=7.5 Hz, H-Bz- 

'^C NMR (CDCI3, Varian VXR 300 MHz, 5): 11.4 (C-18 methyl, q); 13.4 (C-19 
methyl, q); 20.6 (7-0 acetate methyl, q); 21.1 (9-0 acetate methyl, q); 28.0 (C-2, t); 34.1 
(C-6, t); 39.4 (C-3, d); 44.6 (C-14, t); 46.6 (C-8, s); 67.5 (C-10, d); 70.6 (C-7, d); 72.6 (C- 
5, d); 73.2 (C-9, d); 112.2 (C-20, t); 124.0 (C-13, d); 128.4 {C-Bz-ortho, d); 129.6 (C-Bz- 
meta, d); 130.2 (C-Bz-/pso, s); 132.9 (C-Bz-para, d); 134.2 (C-12, s); 145.9 (C-1, s); 
146.4 (C-11, s); 150.4 (C-4, s); 165.2 (CO-Ph, s); 169.9 (CO-Acetate, s); 170.4 (CO- 
Acetate, s). 
Reaction with Iodine and Silver Acetate [4-41 

To a solution of brevifoliol (0.5 g) in benzene (15 ml) were added iodine (0.7 g) 
and silver acetate (0.75 g) and the mixture stirred at room temperature for 20 h. TLC 


showed that the starting material was absent and was replaced by two faster moving 
components. The mixture was filtered and the filtrate washed successively with 
aqueous sodium bisulfite and water and concentrated to dryness. Chromatography on 
silica gel in 4:1 chloroform/ligroin gave the major band, which was obtained as a 
colorless crystalline solid, total yield, 0.12 g; m.p. 250-252 ° C. 

'H NMR (CDCI3, Varian Unity 600 MHz, 6): 1.16, s (H-19); 1.03, s (H-16); 2.08 
(cm, H-14a); 1.27, s (H-17); 2.32 (d, J=13.8 Hz, H-2a); 1.92 (s, methyl, 9-acetate); 1.84 
(m, H-6a); 1.98 (m, H-6p); 2.26 (s, H-18); 2.05 (s, 7-acetate methyl); 3.30 (d, J=13.8 Hz, 
H-2P); 2.28 (cm, H-14p); 2.95, br s (C-15 OH, exchangeable with D2O); 4.38 (t, J=7.2 
Hz, H-13P); 4.28 (br s, H-5p); 4.54 (d, J=13.2 Hz, H-20 B); 4.54 (d, J=13.2 Hz, H-20 B); 
5.51 (dd, J=4.8, 11.4 Hz, H-7a); 6.07 (d, J=10.1 Hz, H-9a); 6.54 (d, J=10.1 Hz, H-10p); 
7.45 (t, J=7.5 Hz, H-Bz-mefa); 7.57 (t, J=7.5 Hz, H-Bz-para); 7.89 (d, J=7.5 Hz, H-Bz- 

^^C NMR (CDCI3, Varian VXR 300 MHz, 5): 13.1 (C-18 methyl, q); 16.1 (C-19 
methyl, q); 20.8 (7-0 acetate methyl, q); 21.5 (9-0 acetate methyl, q); 25.3 (C-17 methyl, 
q); 26.9 (C-16 methyl, q); 31.7 (C-2, t); 38.2 (C-6, t); 142.7(0-3, d); 45.2 (C-8, s); 38.6 
(0-14, t); 65,6 (0-1, s); 67.2 (0-7, d); 76.2 (0-1 0, d); 69.8 (0-5, d); 74.3 (0-15, s); 84.0 
(0-13, d); 72.4 (0-9, d); 64.4 (O-20, t); 128.8 {C-Bz-meta, d); 129.2 (0-Bz-/pso, s); 
129.4(0-Bz-ort/70, d); 133.4 (0-Bz-para, d); 135.5 (0-12, s); 142.7 (0-4, s); 146.4 (0-11, 
s); 164.5 (CO-Ph, s); 169.4 (CO-Acetate, s); 170.3 (CO-Acetate, s). 

FAB-MS (dithiothreotol/dithioerythrotol / TFA, m/z): 577 [M+Na]; 537 [M" NaOH]; 
433 [M'-NaOjOyHs]; 373 [M'-NaOsOyHg -HOAc]; 313 [M'-NaOzOyHs - 2x HOAc]; 253 
[M'-NaOzOyHs-Sx HOAc]. 

OI-MS (methane, m/z): 537.9 [MH' -H2O]; 373.6 [MH^-HsO-HOAc- OgHsOOOH]. 


Acetylation of [4-41 to [4-51 

A sample of [4-4] (.05 g) was acetylated in acetic anhydride (2 ml) and pyridine 
(0.5 ml) at room temperature for 20 h. After addition of water, the solid was filtered and 
crystallized from ether in ligroin, m.p. 250-254° C. 

'H NIVIR (CDCI3, Varian Unity 600 MHz, 5): 1.04 (s, H-16); 1.19 (s, H-19); 1.28 (s, 
H-17); 1.91 (s, methyl, 9-acetate); 1.77 (brdd, H-14a); 2.02 (m, H-6p); 2.05 (s, 7-acetate 
methyl); 2.27 (s, H-18); 2.19 (s, 5-acetate methyl); 2.22 (cm, H-14p); 2.34 (d, J=13.8 Hz, 
H-2a); 3.01 , (br s, C-1 5 OH, exchangeable with D2O); 3.31 (d, J=1 3.8 Hz, H-2(3); 3.90 (d, 
J=13.2 Hz, H-20 B); 4.35 (d, J=13.2 Hz, H-20 A); 4.50 (br m, H-13p); 5.41 (dd, J=3.6, 
13.2 Hz, H-7a); 5.46 (d, J=4.2 Hz, H-5p); 6.04 (d, J=10.1 Hz, H-9a); 6.57 (d, J=10.1 Hz, 
H-10p); 7.45 (t, J=7.5 Hz, H-Bz-mefa); 7.57 (t, J=7.5 Hz, H-Bz-para); 7.89 (d, J=7.5 Hz, 


^^C NMR (CDCI3, Varian VXR 300 MHz, 5): 12.9 (C-18 methyl, q); 16.1 (C-19 
methyl, q); 21.7 (7-0 acetate methyl, q); 21.5 (9-0 acetate methyl, q); 21.7 (5-0 acetate 
methyl, q); 25.3 (C-16 methyl, q); 27.0 (C-17 methyl, q); 31.5 (C-2, t); 32.9 (C-6, t); 38.3 
(C-14, t); 45.2 (C-8, s); 63.5 (C-1, s); 65.4 (C-20, t); 67.6 (C-7, d); 70.3 (C-5, d); 72.3 (C- 
9, d); 74.4 (C-15, s); 76.3 (C-10, d); 83.2 (C-13, d); 127.3 (C-4, s); 128.8 (C-Bz-meta, d); 
129.1 (C-Bz-/pso, s); ^29.4{C-Bz-ortho, d); 133.4 (C-Bz-para, d); 135.5 (C-1 2, s); 
142.7(C-3, d); 145.6 (C-3, s); 147.6 (C-11, s); 164.6 (CO-Ph, s); 169.6 (CO-Acetate, s); 
170.3 (CO-Acetate, s); 170.8 (CO-Acetate, s). 
Reaction with N-Bromosuccinimide and Silver Acetate [4-61 

A solution of brevifoliol (0.2 g) in benzene (10 ml) was stirred with N- 
bromosuccinimide {NBS} (0.125 g, recrystallized from water). After 2 h the starting 
material was absent with two faster moving compounds being present. To the reaction 
mixture was added silver acetate (0.125 g) and stirred for another 2 h. 


At this point, tlie previous major compound moved further to give a new product. 
The mixture was filtered, the filtrate washed with aqueous sodium bisulfite, followed by 
water and concentrated to dryness. The product was chromatographed on a silica 
column in 4:1 chloroform/ ligroin. The major component was obtained as a colorless 
crystalline solid, yield, 0.1 g. The compound was found to be identical with the product 
obtained from the reaction of brevifoliol with iodine and silver acetate. 
Reaction of [4-4] with N-Bromosuccinimide [4-71 

A solution of [4-4] (0.04 g) in benzene (5 ml) was stirred with N- 
bromosuccinimide (25 mg) at room temperature. After 2 h, TLC showed formation of a 
slightly faster moving compound, which was separable from the starting material only 
after 2 or 3 developments of the TLC plate. The reaction mixture was washed with 
aqueous sodium bisulfite, followed by water and concentrated to dryness. The product 
was crystallized from ether in ligroin, m.p. 185-188 °C. 


As discussed in Chapter 3, the yield of taxol from the bark of Taxus brevifolia by 
using the conventional methods of isolation was of the order of 0.01%. It was also 
shown that through the use of these same methods, no other useful analogues could be 
isolated in any significant yields. As a consequence of these results and strong 
ecological considerations, an intense search was started with the aim of finding a source 
that is renewable, and which can match the bark in the yield of taxol. Many of the 
available species of Taxus, as well as the various parts of these plants were examined 
through the use of analytical high performance liquid chromatography (HPLC) and thin 
layer chromatography (TLC). These searches led to the selection of the needles of the 
ornamental yew, Taxus x media Hicksii as a possible answer to the problem. The 
ornamental yew is capable of being grown in a nursery type setting, and on a large 
scale, so that the needles may be clipped twice a year, and the taxol, which is found to 
be present to the extent of 0.01% be isolated from them. 

At the time of this research (1992-93), almost all of the studies carried out on this 
species consisted of HPLC analyses. Other than the isolation of taxol by the standard 
procedure with a total yield of 0.006%, no information had been published either on the 
taxane constituents, or even a method for the practical isolation of them. In these HPLC 
analyses, it was recognized that in the extracts of the ornamental yew, taxol was 
accompanied by other co-eluting taxanes and these could contribute some errors in the 
total yield calculations. These co-eluting taxanes were isolated in minute yields, in the 
form of two components (0.8 mg and 1.2 mg); each representing an equilibrium mixture 



of two components. On the basis of NMR spectral evidence, structures were assigned 
to tinese two components (Castor & Tyler, 1993). 

Due to tine presence of pigments, waxes and other impurities, the isolation of 
taxol and other taxanes from the needles was expected to be more difficult when 
compared to their isolation from the bark of T. brevifolla. A project was started in this 
laboratory to meet the need for a practical method for the isolation of taxol and other 
related taxanes from the bark and needles of various Taxus species in spite of these 
challenges. The application of a preparative scale reverse phase column 
chromatography technique proved to be surprisingly successful in the processing of the 
extracts of T. brevifolia. 

To begin with, the HPLC analysis of the extract of the needles of the ornamental 
yew, as shown in Figure 5-2, clearly shows that taxol is accompanied by several major 
taxane components, which are present in much higher concentrations than taxol itself. 
In view of such relatively high concentrations of these components, it is surprising that 
only such minute amounts of two of these mixtures could be isolated earlier, as indicated 
above. Also, no other characterizing data were provided other than the spectral data. 
This laboratory's objective was the development of a simpler procedure for the isolation 
of taxol with potential for large-scale use, in addition to more fully characterizing the 
major taxanes present in the extract. The needles of the ornamental yew (200 lbs., 
dried) were received through the courtesy of Hauser Company, Boulder, CO, during 
May-June 1993. 

The extraction was carried out three or four times using methanol and the extract 
concentrated to a syrup. The resulting concentrate was then partitioned between water 
and chloroform, and the organic layer containing the taxane fractions was concentrated 
to a thick semi-solid mass, which was used directly in the next step. 


The reverse phase column procedure was earned out similar to what was used 
with the needle extract of T. brevifolia, as described in Chapter 3. Approximately 200 g. 
of the chloroform extract was dissolved in acetonitrile (see experimental) and stirred with 
the equilibrated C-18 bonded silica. This slurry was then diluted to the appropriate 
concentration of the acetonitrile and the added to the column prepared from 800 g of the 
C-18 silica. Elution was carried out using a step gradient: 30, 35, 40, 45, 50 and 60% 
acetonitrile in water, and the eluate collected in fractions of 200 ml. As was seen in the 
case of the columns on the bark extract of T. brevifolia, when the fractions remained at 
room temperature for about a week, crystals began to separate from the fractions in 
different regions of the elution. These were filtered and further purified by either 
recrystallization or a small column of normal phase silica where necessary. 

The progress of elution of the column is shown in Figure 5-3. As anticipated, 
taxol was accompanied by two other taxanes, which were present in higher 
concentrations than taxol. However, all of these crystallized out of the fractions. 

The early fractions contained the bulk of the UV absorbance, and from these 
could be isolated a crystalline solid, which was a non-taxane compound. The next major 
component that emerged with the 35-40% acetonitrile in water was shown to be 
brevifoliol as described in Chapter 3. With the 45-50% acetonitrile and water solvent 
were eluted taxane 1, taxane II, followed by taxol, all of which crystallized from their 
respective fractions, with some overlap. 

The column was finally washed with a mixture of methanol and ethyl 
acetate/ligroin (2:1:1) which stripped the column of all the waxes, chlorophylls and other 
pigments. After, washing with methanol, followed by 25% acetonitrile and water the 
column was made ready for another run. Figure 5-1 shows the steps involved in the 
fractionation of the extract of Taxus x media Hicksii. 




Dried Needles of 
Tax us X media Hicks I i 



"Extract Solids" 

Reverse Phase Column 

Filter Crystals 

Recrystallize or chromatograph 

Brevifoliol Taxanes I and II Taxol 


Taxane IV 



Figure 5-1 : Fractionation of the Extract of Taxus x media Hicksii Needles 



Taxus floridana 

Taxus X media Hicksii 

Figure 5-2 : HPLC Trace of Taxanes Coeluting with Taxol. 

Column Elution, Absorbance vs. Time 








I. ii.--.iii-irMi-.iii 



Figure 5-3 : Progress of Elution of Taxanes from Reverse Phase Column. 


The reverse phase column run on 600 g of the extract obtained from 12 Kg of the 
dried needles was applied to a column prepared from 3 Kg of the C-18 bonded silica. 
The yields of the products obtained were good and important values are listed below. 
Brevifoliol [3-11 

The fractions containing this component were combined, concentrated to dryness 
and chromatographed on a normal phase silica column. The major component was 
obtained as a colorless crystalline solid, which was found to be identical with brevifoliol 
[3-1] on the basis of its spectral data. 
Taxanes I [5-11 and II [5-21 

Additional chromatography of the mixture of the taxanes I and II and taxol on a 
normal phase silica column gave some separation of the two taxanes. Although they 
could be further separated and obtained as crystalline solids, they still represented an 
equilibrium mixture, as was indicated by the ^H- and "C NMR spectra of the individual 
crystalline samples. From the spectral data, these two were identified as a mixture of 
5-O-cinnamoyl-10-acetyl taxicin I [5-1] and 5-0-cinnamoyl-9-acetyl taxicin I [5-2], which 
were isolated (Chmurney ef al. 1993) from the needles of T. x media Hicksii and from the 
needles of T. baccata (Appendino ef al. 1992). The former authors obtained them in 
quantities not sufficient for physical properties, and the latter authors obtained them as 
amorphous powders, by using HPLC and preparative TLC. 

The mixture of [5-1] and [5-2] on acetylation with acetic anhydride and pyridine 
gave the triacetate [2-1], which was obtained as a crystalline solid and was found to be a 
single entity unlike the starting material. It was also identical with the taxane III (see 



The crude crystalline solid obtained from the reverse phase column was 
recrystallized. Its spectral and analytical data agreed with those given for 5-0- 
cinnamoyl-2a,9a, 10p-triacetyl taxicin I (Appendino et al. 1993; Baxter ef a/. 1962). 

Taxane IV [2-21 

This was also purified by recrystallization of the crude crystals obtained directly 
from the fractions. It was found to be identical with 5-0-cinnamoyl 2a, 9a, 10|3-triacetyl 
taxicin II, described by Appendino et al. (1992) and Baxter et al (1962). 
Taxol [5-31 

The chromatography using normal phase silica column as described under 
taxanes I and II yielded taxol, which was purified by crystallization. The sample was still 
contaminated with some of the taxanes I and II. For complete purification, the mixture 
was subjected to ozonolysis which converted these two taxanes to more polar 
compounds from which taxol could be readily separated and obtained pure. Using this 
method, taxol was obtained in a yield of 0.015% based on the dry needles. This was 
significantly better than the reported yield of 0.006% (Witherup et al. 1990). 
Ozonolysis of [2-21 

Because of the presence of the cinnamoyi ester function in compounds [5-1], 
[5-2], [2-1] and [2-2], they all undergo ozonolysis. This method gives a convenient way 
of separating taxanes [5-1] and [5-2] from taxol, with which they co-elute. In order to 
determine the nature of the product of ozonolysis, taxane [2-2] was subjected to this 
reaction and the product recovered and obtained as a crystalline solid. Its NMR spectral 
characteristics indicated a hydrated aldehyde with the structure shown in [5-6]. 


Thus, in summary, dried needles of Taxus x media Hicksii were extracted and 
the total chloroform extract applied to a C-18 reverse phase column. A number of 
components were separated, such as brevifoliol, taxanes l-IV and taxol, which 
crystallized out directly from the fractions. Separation of taxol from taxanes I and II 
could be carried out directly by ozonolysis of the mixture, followed by chromatography 
on either a normal phase or reverse phase silica column. 



Dried needles of Taxus x media Hicksii (50 lbs) were extracted with methanol as 
described in Chapter 3. The combined concentrate was partitioned between water and 
chloroform (10 gallons each). The organic layer was separated and the extraction 
repeated twice more using 5 and 3 gallons of the solvent, respectively. 

The combined chloroform layers were concentrated under reduced pressure to 
yield a dark green semi-solid, representing approximately 5% of the weight of the dried 

Approximately 800 g of C-18 bonded silica gel was poured into a glass Michell- 
Miller type column (2.5 x 24") using methanol (Ace Glass, 1430 North West Blvd., 
Vineland, NJ 08360). The column was equilibrated with 25% acetonitrile in water. The 
chloroform extract solids (200 g) was dissolved in acetonitrile (400 ml) in a 4 L stainless 
steel beaker, by warming in a hot water bath. To this was added approximately 200 g 
equivalent of the equilibrated C-18 bonded silica (20-25% of the column material). While 
the mixture is being stirred vigorously, 25% acetonitrile and water 500 ml was added, 
followed by water (approximately 800 ml). After stirring for 15 min. it was checked for 




uniformity of tine slurry and the absence of oily or waxy material, or lumps. The slurry 
was filtered under gentle suction and the solid was resuspended in approximately 500 ml 
of the filtrate to give a thin enough slurry for pouring. It was added to the column, the 
container rinsed and the rinse transferred to the column. 

The remainder of the filtrate was pumped onto the column using a metering 
pump (Pulsa 680, Pulsafeeder Inc., Rochester, NY). After the sample addition was 
completed, elution was started using 30% acetonitrile and water. This was followed by 
35, 40, 45, 50 and 60% acetonitrile and water. The column was then washed with 100% 
methanol. Final washing of the column with a mixture of methanol and ethyl acetate and 
ligroin removed the green pigments and other lipid-soluble components. 

Fractions of 200 ml volume were collected and monitored by UV absorbance, 
TLC and analytical HPLC. After this, those fractions that contained significant UV 
absorbance and/ or components detectable by TLC or HPLC were set aside for 7-10 
days, whereby crystals began to appear from a number of fractions. These were filtered 
in groups, characterized and treated appropriately, as described below. 

Characterization of the Taxane Components of Taxus x Media Hicksii 

Brevifoliol [2-11 

Fractions from the 40% acetonitrile and water were concentrated to dryness, the 
solid taken up in chloroform and applied to a column of normal phase silica (40 g). 
Elution with 2-5% acetone in chloroform gave the major band. The fractions that contain 
this component were combined, concentrated to dryness and the solid crystallized from 
acetone in ligroin. The crystalline product, yield, 0.8 g (0.02%) m.p. 220-222 ° C was 
found to be identical on the basis of NMR spectral data with brevifoliol described in 
Chapter 3. 


Taxanes I and II [5-1] and [5-2] 

The crude crystals that separated out from the fractions (8 g) consisting of [5-1], 
[5-2] and taxol [5-3] were processed by two methods. In one, the mixture (4 g) was 
taken up in chloroform and ligroin (3:1, 50 ml) and applied to a column of normal phase 
silica (60 g). The mobile phase was successively changed to chloroform, 2% acetone, 
5% acetone, 2% methanol and 5% methanol in chloroform. Compounds [5-1] and [5-2] 
appeared in the 2-5% acetone and chloroform eluate partially separating from each 
other. Continuing with 2% methanol in chloroform gave taxol with small amounts of [5-1] 
and [5-2]. 

To obtain further purification of [5-1] and [5-2] the mixture was taken up in 40% 
acetonitrile and water and applied to a column of C-18 bonded silica. The column was 
eluted with 45 and 50% acetonitrile and water. As the fractions from the 45% acetonitrile 
and water elution stood for about a week, crystals appeared over a range of tubes and 
these were filtered in groups. Although [5-1] and [5-2] were separated, such that each 
contained the other to the extent of 10% or less, recrystallization gave worse mixtures, 
thus suggesting that isomerization (or equilibration) was taking place during the process. 
Data obtained on a crystalline (9:1 mixture of [5-1] and [5-2]: m.p. 136-138° C, [a]D^^ 
+214° (c 1.04, CHCI3); (lit. Appendino et al. 1992 on an amorphous sample, m.p. 163- 
165° C and [a]D^' +185°). 

Analysis calculated for CsiHsgOg, H2O: C, 66.89; H, 7.24. Found: C, 66.51; H, 


The ^H- and ^^C NMR spectra of the crystalline [5-1] and [5-2] gave evidence of 
mixtures of two compounds. From the spectral data, these two were identified as a 
mixture of 5-0-cinnamoyl-9-acetyl taxicin I [5-1] and 5-O-cinnamoyl-10-acetyl taxicin I [5- 
2] described by Chmurney et al. (1993) from Taxus x media Hicksii and by Appendino et 


al. (1992) from Taxus baccata. The former authors isolated insufficient amounts for 
characterization and the latter authors obtained them as amorphous powders by using 
HPLC and preparative TLC. 

The mixture of [5-1] and [5-2] on acetylation with acetic anhydride and pyridine 
gave the acetate, readily obtained as a crystalline solid, m.p. 238-241° C, the NMR 
spectrum of which showed that it was a single entity, unlike the starting material. It was 
also identical with taxane III (see below). 
Taxanelll [2-1] 

The crude crystals of taxane III obtained from the fractions with 50% acetonitrile 
and water were filtered and recrystallized from acetone in ligroin to obtain colorless 
needles, yield, 0.8 g (0.02%); m.p. 238-241° C, [a]D'' +214 (CHCI3, c 1.04); (lit. +218, 
Baxter, 1962); FAB-MS (m/z): 645 (M' +Na); 623 (M* + H); 475 [(MH)' - 148 
(cinnamoyi)], 415 (475-AcOH); 355 (415-AcOH); 295 (355 - AcOH). The spectral data 
showed that it is the 5-0-cinnamoyl-2a, 9a, 10p-triacetyl taxicin I (Appendino et a/. 1992; 
Baxter ef a/. 1962). 

Analysis calculated for C35H42O10, H2O: C, 65.61; H, 6.92. Found: C, 66.00; H, 
Taxane IV [2-2] 

This compound also crystallized out directly from the fractions. The crude 
crystals were purified by recrystallization from acetone and ligroin, yield, 0.8 g (0.02%); 
m.p. 265-267° C; [a]D'' +133°(C 0.98, CHCI3); (lit. +137, Baxter et al. 1962); FAB-MS: 
607 (MH^); 459 (607 - 148 (cinnamate)); 399 (459 - HOAc); 339 (399 - HOAc); 279 (339 
- HOAc). 

Analysis calculated for C35H42O9: C, 69.02; H, 7.03. Found: C, 69.29, H, 6.98. 


The analytical and spectral data of [2-2] indicated that it was identical with 5-0- 
cinnamoyl taxicin II: 2a,9a,10p-thacetate described by Appendino et al. (1992) and 
Baxter ef a/. (1962). 
Taxol [5-31 

In the silica column described above under the purification of connpounds [5-1] 
and [5-2], taxol (approximately 0.8 g) was eluted by 2-5% methanol in chloroform. A 
small portion was crystallized from acetone and ligroin to obtain colorless needles of 
taxol. The ^H NMR spectrum showed that the compound still had appreciable quantities 
of compounds [5-1] and [5-2]. To remove these compounds completely, ozonization 
was carried out on the rest of the sample in chloroform and methanol (9:1, 30 ml) at -70° 
C for 10-15 min. The reaction mixture was treated with dimethyl sulfide (0.5 ml) and let 
stand at room temperature for 2 h. 

After concentration to dryness, the sample was chromatographed on normal 
phase silica (25 g) in chloroform. Elution with 2% methanol in CHCb gave taxol which 
was crystallized from ligroin to obtain pure taxol, free from compounds [5-1] and [5-2], 
yield, 0.5 g (0.012%). Its spectral properties agreed with those of an authentic sample. 

Alternatively, the crude crystalline solid consisting of compounds [5-1] ,[5-2] and 
taxol was directly ozonized in chloroform and methanol as before (but without he 
intermediate silica column purification). After decomposition of the ozonide, and 
concentration, the sample was subjected to chromatography and taxol isolated from the 
column. It was crystallized as before to yield 0.75 g (0.015%). The products of 
ozonization of compounds [5-1] and [5-2] were more polar than the original compounds 
and separated from taxol in the normal phase silica column. 


Ozonolysis of Compound [2-2] 

A solution of compound [2-2] (1 g.) in chloroform and methanol (30 ml, 9:1) was 
cooled in a dry ice and acetone bath and saturated with ozone for 10-15 min. TLC 
showed that the starting material was absent and ozonide being formed (detected by 
spraying with starch and potassium iodide which gave a blue color). After the 
decomposition of the ozonide by dimethyl sulfide, the reaction mixture was washed with 
water and concentrated to dryness. The product was crystallized from acetone in ligroin 
to obtain colorless needles, yield, 0.8 g, m.p. 168-170° C, [a]D^^ +130 (c 1.06, pyridine); 
HRMS: 569.2239, Calc. for C27H36O13, 569.2234. 


Taxus floridana is a species of Taxus, native to Florida. Its distribution is said to 
be limited to a small area along the Apalachicola River. It is a shrub and used frequently 
as an ornamental plant. As it is so with the other species of Taxus, the leaves of T. 
floridana are also reputed to be toxic to livestock and humans. 

During the intensive search to find alternative sources for taxol to replace the 
bark of the Pacific yew, many species of Taxus from the United States, Canada, Europe 
and Asia were examined. However, there was no study of the taxane constituents of 
Taxus floridana. Our laboratory undertook this task to evaluate its usefulness as a 
possible source for taxol. 

There was also an impetus for this study from another source. In exploring 
alternative sources to replace the bark of the Pacific yew, the National Cancer Institute 
(NCI) was interested in knowing whether the Taxus plants can be grown under 
hydroponic conditions, as opposed to their growing in their natural state. If these plants 
can be so grown under hydroponic conditions, which will eliminate the problem of having 
to harvest the tree bark, the next question was whether they produce taxol in adequate 
yields. Accordingly, the NCI approached our laboratory, and that of Prof. George 
Hochmuth Jr. of IFAS, University of Florida, to study this aspect. The hydroponic 
cultural techniques were studied by the IFAS laboratory and the isolation and 
characterization of taxanes by our laboratory. It was soon found that the two most well- 
known species of Taxus, namely T brevifolia and T baccata could not be readily 
propagated under normal hydroponic conditions, because their growth rate was very 



slow. However, T. floridana responded satisfactorily and could be propagated under 
available conditions. This species was therefore studied in our laboratory for its taxane 

The needles of T. floridana were collected from the campus and were extracted 
without drying. After extraction with methanol as before, concentration to remove the 
solvent, and partition between chloroform and water, the organic layer was concentrated 
to a dark green semisolid. Fractionation was again carried out using the reverse phase 
column techniques as was described under the needles of the other Taxus species in 
Chapters 3 and 5. 

The crude chloroform extract was first tested by analytical HPLC to see the 
elution pattern of the taxane constituents. Taxol was clearly recognizable at its normal 
location, and in contrast to the observation with the needles of Taxus x media Hicksii, 
where there were co-eluting taxanes, the taxol from the extract of the needles of T. 
floridana was relatively free from such interfering taxanes. There were other taxanes 
situated at different locations. 

Elution of the reverse phase column was carried out using a step gradient of 30, 
35, 40, 45, 50 and 60% acetonitrile/ water. When the fractions were let stand at room 
temperature for 3-5 days, taxol and several other taxanes crystallized out as before. 

The initial eluates from the column from 25-30% acetonitrile/ water contained 
highly polar phenolic constituents. The first taxane component to appear from the 
reverse phase column emerged with the 30-35% acetonitrile/water solvent, and 
crystallized almost immediately. This was found to be 10-deacetyl baccatin III [2-7]. The 
next taxane was eluted with the 40% acetonitrile/ water and it was found to be identical 
with brevifoliol [3-1]. With the 45% acetonirile/ water, was eluted another crystalline 
compound which was found to be a new compound, and was named taxiflorine [6-1]. 
Continued elution with 50% acetonitrile/ water gave two crystalline compounds in 


succession. One of these was identified as baccatin VI [6-2], and the second one was 
taxol [5-3]. 


Taxiflorine [6-1] was readily obtained as a colorless crystalline solid. Its 
elemental analysis agreed with the molecular formula C35H44O13. Its ^H NMR spectrum 
in CDCI3 showed broad and rounded peaks with poor resolution. In DMSO-de, the 
spectrum gave sharper signals but showed double the number of peaks in certain 
positions. The ^^C spectrum also exhibited extra peaks, which suggested that the 
compound was a mixture of rotamers in equilibrium. One could infer the presence of 
ester functions from the spectra, with four acetates and one benzoate, and an oxetane 

Acetylation of taxiflorine gave a monoacetate [6-3], which gave sharp signals in 
its ^H NMR spectrum, with the expected number of peaks, thus showing that it is a single 
compound, unlike the starting material. Although the acetate was isomeric with baccatin 
VI, it was different. The most striking difference between the two spectra was seen with 
the signal for the H-13. In the acetate of taxiflorine, this signal was at 5 5.60, while the 
same was found at 5 6.3 in baccatin VI. A comparison with other related taxanes 
showed that in those with the 6-membered A-ring, the H-13 signal appears at 5 6.2-6.5, 
whereas in taxanes with a 5-membered A-ring, as in the 11(15-»1)-abeotaxanes, it 
appears at 5 5.4-5.7 (Appendino et al. 1993B). 

Positions 9 and 10 in taxiflorine carry a free hydroxyl and a benzoate function. 
To locate the benzoate, a comparison of the signals due to H-9 and H-10 in taxiflorine 
were compared with the corresponding signals in the monoacetate. With the two signals 
at 5 6.30 and 5 5.90 in taxiflorine, the latter undergoes a down-field shift from 5 5.90 to 


5 6-20, whereas the peak at 8 6.30 remains essentially unchanged (5 6.37). With the 
reasoning that the allylic H-10 must be more down-field than H-9, the signal at 8 6.30 
may be assigned to H-9 and the one at 8 5.90 for the H-10. This leads to the 
assignment of the structure of taxiflorine as [6-1], with the hydroxyl at 9 and the 
benzoate at 10. Based on the ^H,^H-COSY and ^H.^^C-HETCOR spectral data, the four 
acetate functions were assigned as 2a, 4a, 7p and 13a. 

Benzoylation of [6-1] was carried out to yield the monobenzoate [6-4], which also 
gave a ^H NMR spectrum that indicated that it was a single entity. Taxiflorine was also 
saponified to the heptaol and re-acetylated to the hexa-acetate [6-5]. The ^H NMR 
spectra of [6-3], [6-4] and [6-5] are shown in Table 6-1. 

The structure of [6-1] with the hydroxyl at C-10 and benzoate at C-9 has the 
potential for intramolecular transesterification occurring between the 9-benzoate, as well 
as the 7-acetate (Lewis et al. 1993). The fact that the monoacetate and the 
monobenzoate gave single products discounted the possibility that transesterification 
was responsible for the anomalous NMR spectra of [6-1], To verify if the appearance of 
the spectrum is due to an rotamehc equilibrium, the spectrum was taken in DMSO-de at 
temperatures between -20° and 100°. At lower temperatures, the spectra were sharper 
and showed two sets of peaks for some protons. At higher temperatures, the peaks 
coalesced into a single set, as well as became broad to the extent that they were barely 
seen. This behavior suggested that the conformational equilibrium between the 
rotamers is responsible and that the presence of the 10-OH facilitates this process. As 
indicated earlier, during the exploration of possible alternative sources for taxol to 
replace the bark of the Pacific yew, scant, if any attention was paid to Taxus floridana. 
The species T. x media Hicksii (the ornamental yew) was selected as a possible source 
for taxanes in the future. 


Table 6-1 : Proton NMR Spectra of Compounds [6-3], [6-4] and [6-5]. 


Compound [6-3] 

Compound [6-4] 

Compound [6-5] 


6.19, d, J=7.8Hz 

6.26, d, J=7.8 Hz 

6.07, d, J=7.8 Hz 


2.99, d, J=7.8 Hz 

3.06, d, J=7.8 Hz 

2.92, d, J=7.8 Hz 


4.98, d, J=7.5 Hz 

5.01,d, J=7.5Hz 

4.98, d, J=7.5 Hz 


2.68, m 

2.70, m 

2.52, m 


1.84, m 

1.84, m 

1.84, m 


5.52, m 

5.64, m 

5.49, t, J=7.8 Hz 


6.32, d, J=10.8HZ 

6.48, d, J=10.8Hz 

6.04, d, J=10.8Hz 


6.37, d, J=10.8Hz 

6.72, d, J=10.8Hz 

6.27, d, J=10.8, Hz 


5.62, t, J=7.8Hz 

5.64, m 

5.61,t, J=7.8Hz 


2.30, dd,J=7.4,14.2Hz 

2.34, dd. 

2.30, m 


1.72, dd,J=7.4,14.2Hz 

1.78, m 

1.72, m 


1.16, s 

1.24, s 

1.15, s 



1.21, s 



1.72, s 

1.72, s 

1.83, s 


1.64, s 

1.95, s 

1.66, s 


4.50, 4.42, d, J=7.9 Hz 

4.52, 4.44, d, J=7.2 



7.93, d 




7.45, t 

7.24, mm 



7.62, t 

7.37, m 



7.63, d, J=7.2Hz 



7.24, m 




7.37, m 



2.14, s 



2.14, s, (2x) 

2.05, s 

2.10, s 


1.86, s 


2.08, s 


1.80, s 

2.03, s 



2.01, s 



1.95, s 

^H NMR were recorded at 600 MHz in CDCI3 on a Varian Unity 600 spectrometer 
at ambient temperature. Cinemical shifts 5 (ppm) are reported relative to TMS as an 
internal standard. 



[6-1] - Taxiflorine R^ = H R2 = CeHgCO 

[6-3] - Taxiflorine Acetate Ri = CH3CO R2 = C6H5CO 
[6-4] - Taxiflorine Benzoate Ri = R2 = CeHsCO 
[6-5] - Hexa-Acetate Ri = R2 = CH3CO 




[6-2] -BaccatinVI 

Figure 6-1 : Taxanes and Analogues from Taxus x media Hicksii 


From the present studies, which compared the taxane composition of both 
species, it became clear that T. floridana would have been a much better choice, for the 
reasons given below: 

1 . Taxol is isolated more easily from the Florida yew than from the ornamental yew, 
because, in the former there are no co-eluting taxanes to interfere and which have to 
be removed as an additional step. The yields of taxol (0.01% from fresh leaves) are 
accordingly better with the Florida yew, than from that of the ornamental yew 
(0.015% from dried leaves). 

2. Besides taxol, the Florida yew gives relatively high yields (0.05-0.06% from fresh 
leaves) of 10-deacetyl baccatin III, which is the most commonly used intermediate for 
the semi-synthesis of taxol. This compound is present in the ornamental yew in 
exceedingly low concentrations. 

3. The other components that contain the oxetane ring, such as baccatin VI and 
taxiflorine, are also useful compounds for the synthesis of taxol analogues. In 
contrast, the taxanes from the ornamental yew that are 1 1 ,4/20 diene taxanes have 
no currently documented use. 

The work described here is the first definitive investigation of the taxane 
constituents of Taxus floridana on a preparative scale giving the actual recovered yields 
of the crystalline compounds. 



The needles and small twigs of Taxus floridana were collected from several 
bushes growing at different locations of the campus of the University of Florida. 
Likewise, they were made available from the plants growing under hydroponic 


conditions. For convenience, they were extracted in fresh state. Several batches were 
collected from the plants on the campus ranging from 1-20 Kg. With the hydroponically 
grown plants, the smallest amount was 54 g of the fresh needles, and the largest, 2.5 
Kg. The method of extraction, concentration, partition between water and chloroform 
were the same as was described in Chapter 3. The yield of the chloroform extract varied 
from 20-25 g per Kg of the fresh leaves and twigs. 

Before the conditions for the reverse phase column chromatography were fully 
developed, an alternative procedure was tested for the purpose of making the sample 
preparation easier. The chloroform extract, especially of the needles, usually contains a 
higher amount of waxes, chlorophylls and other lipid-soluble components and can 
potentially pose problems in the preparation of the sample for applying to the column. 
For this reason, a study was made to see at what concentration of methanol or 
acetonitrile would be needed to obtain an essentially clear solution that can be applied to 
the column. 

It was found that at least an 80% methanol in water would be necessary to 
prepare a 10% solution of the chloroform extract solids. However, at this concentration 
of the solvent, taxol and most of the other taxanes do not remain on the column. It was 
also found that if such a solution is passed through a column of C-18 bonded silica, 
almost all of the chlorophylls, waxes etc., remain on the column, while taxol and other 
taxanes appear in the effluent and washes. There was also a reduction of the solids 
content by 50-60%, which meant that the waxes and other lipid-soluble components 
account for this much of the chloroform extract and can be readily removed from the 


The material obtained by concentrating the effluent and washes was much less 
difficult to prepare as the slurried sample for applying to the column. In order to carry 
out this wax-removal operation, it was only necessary to use a ratio of 3 g of the C-18 


silica for 1 g of the extract. The chlorophylls and waxes that were held up on the column 
could be readily removed by washing with a mixture or methanol/ ethyl acetate/ ligroin 
(2:1:1). Examination by TLC showed that this wash did not contain any taxane 
constituents. This procedure was not used in later trials, as methods were found for a 
successful and convenient preparation of the sample slurry made it unnecessary, as 
described in Chapter 3. 

Characterization of the Taxane Constituents of Taxus floridana 

The results given here represent the work carried out on a 20-Kg batch of the 
fresh needles. 
10-Deacetvl Baccatin 111 [2-71: 

Elution with 30% acetonitrile in water gave this component which crystallized 
almost immediately. After a week, the crystals were filtered off, dried and recrystallized 
from methanol/ chloroform, yield, 12 g (0.06%); m.p. 232-234° C. The spectral 
properties were identical with those described in the literature (Chauviere et al. 1981, 
Appendino ef a/. 1993b). 
Brevifoliol [3-11: 

Fractions from the 35-40% acetonitrile eluate, which contained this component 
but did not give a crystalline solid directly, were combined, concentrated to dryness and 
the solid (3 g) was applied to a normal phase silica column (120 g) in chloroform. 
Elution with 2% methanol in chloroform gave the major band, the fractions from which 
were combined, concentrated and the solid crystallized from acetone / ligroin to give 1 g 
of [3-1], m.p. 220-222° C. Its spectral data proved to be identical with those described in 
Chapter 3. 


Taxiflorine [6-11: 

The crude crystalline solid (2.5 g) that separated from the fractions from 45% 
acetonltrile/ water was filtered and purified by recrystallization from acetone/ ligroin to 
give [6-1] as a colorless crystalline solid, yield, 1.2 g (0.006%); m.p. 254-255° C, [alo'' 


Analysis calculated for C35H44O13: C, 62.48; H, 6.59. Found: C, 62.12; H, 6.63. 
Baccatin VI [6-21: 

Eluates from the 50% acetonitrile/ water gave crystals in a number of fractions. 
These were filtered into groups and tested by TLC and analytical HPLC. The earlier 
fractions contained mostly baccatin VI, with gradually increasing amounts of a slower 
compound, shown to be taxol. The crystals from the first group containing mostly [6-3] 
(3.5 g) were dissolved in chloroform (50 ml) and passed through a column of Florisil (20 
g) for the purpose of decolorization. The effluents and washes were combined, 
concentrated to dryness and the solid crystallized from acetone/ ligroin to give pure [6-3] 
yield, 1.6 g. Together with the amount obtained from the next fraction, the total yield 
was 1.95 g (0.01%); m.p. 250-252 ° C (lit. 248-250 ° C decomp., Senilh et al. 1984); 
[a]o23 .i-|o (chloroform, c 0.98) (lit. -5, chloroform, c 1.3, Senilh et al. 1984); MS(FAB); 

737 [M+Na]", 697 [M-H2O]". 

^H NMR (CDCI3, 300 MHz, 5): 1.22 (17-Me, s); 1.60 (19-Me, s); 1.78 (16-Me, s); 
1.87 (6a, cm); 1.99 (OAc-Me, s); 2.02 (OAc-Me, s); 2.04 (C-14p, unresolved mult.); 2.10 
(18-Me, s); 2.10 (OAc-Me, s); 2.19 (OAc-Me, s); 2.20 (C-14a, unresolved mult.); 2.28 
(OAc-Me, s); 2.50 (C-6p, cm); 3.18 (C-3, d, 6 Hz); 4.13 (C-20, d, 8.4 Hz); 4.34 (C-20, d, 
8.4 Hz); 4.97 (C-5, d, 8.4 Hz); 5.55 (C-7, dd, 7.5, 9.3 Hz); 5.87 (C-2, d, 6.0 Hz); 5.99 (C- 
9, d, 11.1 Hz); 6.17 (C-13, dd, 7.6, 9.3 Hz); 6.22 (C-10, d, 11.1 Hz): 7.48 (Ar-meta, t, 7.8 
Hz); 7.61 (Ar-para, t, 7.5 Hz); 8.09 (Ar-ortho, dd, 7.2, 1,3 Hz). 






■"^ CD 
1 1 


1 1 1 1 1 1 1 1 1 


O ^ 



in ^ 



m ^ 


h- CD CO CO n CO LO 

I I I I I I I I I I I I 

r^-^i~--coc7)i— r-oocDCMh--h-; 

O O O O O O 
O O O O O O 


cr o 

O O O O O N 

< < < < < CD 

1 1 1 1 1 1 







N N 

m CD 

1 1 



Tj- T- 00 t- CO CO 





O O en (J) CO CD 
r-- h- CD CD CD CD 




cj) a> 




















































^^C NMR (CDCI3, 300.075 MHz, 5): 75.0 (C-1); 78.8 (C-2); 47.5 (C-3); 81.6 (C-4); 
83.8 (C-5); 34.3 (C-6); 73.3 (C-7); 45.8 (C-8); 71.9 (C-9); 69.7 (C-10); 133.5 (C-11); 
142.0 (C-12); 70.4 (C-13); 35.3 (C-14); 42.8 (C-15); 22.4 (C-16); 28.3 (C-17); 12.9 (C- 
18); 15.0 (C-19); 76.5 (C-20); 20.7; 20.9, 21.2, 21.4, 22.7 (5x OAc Me); 168.6, 168.9, 
169.6, 169.9, 170.2 (5X OAc CO); 166.8 (Bz CO); 129.2 (Bz ipso); 128.5 (Bz ortho); 
129.9 (Bz meta); 133.5 (Bz para). 

Analysis calculated for C37H46O14: C, 62.18; H, 6.49. Found: C, 61.83; H, 6.45. 
Taxol [5-3]: 

Crystals (4.5 g) from the second part of the peak which contained mostly taxol. 
were combined dissolved in chloroform (60 ml) and chromatographed on Florisil(40 g). 
Elution with chloroform gave more of [6-3] and subsequent elution with 5% acetone in 
chloroform gave taxol, which was recovered by concentration of the appropriate fractions 
and crystallized from acetone and ligroin to obtain taxol, yield, 1.98 g (0.01%); m.p. 220- 
222 ° C. The spectral and chromatographic properties of the sample agreed with those 
of taxol. 
Acetvlation of Taxiflorine to [6-3]: 

An aliquot of [6-1] (0.05 g) was acetylated by acetic anhydride (2 ml) and pyridine 
(0.5 ml) at room temperature for 16 h. Water was added, the solid filtered and purified 
by chromatography on a silica column using chloroform / ligroin (2:1) to obtain [6-3] as a 
white powder. 
Benzovlation of Taxiflorine to [6-4]: 

To a solution of [6-1] (0.05 g) in pyridine (2 ml) was added dropwise with stirring 
at 0-5° C, benzoyl chloride (0.1 ml). After 20 h water was added followed by 2N sulfuric 
acid and the solid filtered. The product was purified by chromatography as given under 
[6-3], to obtain [6-4] as a white powder. 


Saponification and Acetylation of [6-1] to [6-5]: 

A solution of taxiflorine (0.1 g) in methanol (5 ml) was treated with 2N potassium 
hydroxide (1 ml) and the mixture let stand at room temperature for 2 h. TLC showed that 
the starting material was no longer present, along with the appearance of a very slow 
moving component. The solution was acidified and extracted with chloroform (3x) and 
the combined extracts concentrated to dryness. The residue was dissolved in acetic 
anhydride (2 ml) and pyridine (0.5 ml) and let stand for 16 h. Addition of water, filtration 
of the solid and chromatography on a normal phase silica gave [6-5] as a white powder. 



Some of the benefits of using reverse phase rather than normal phase 
chromatography have been described in previous chapters. Two important 
disadvantages of normal phase silica gel chromatography are the acidic nature of silica 

I and the tendency for irreversible adsorption to occur. Both of these problems can lead 

to significant loss of the compound(s) of interest. Fortunately, almost all free acidic 

i groups are capped during the bonding process used to make reverse phase silica, 

followed by a final capping with trimethylsilyl groups. This process effectively eliminates 
these problems of acidity and irreversible adsorption. 

These properties allow recovery of many compounds that would normally not be 
amenable to silica gel chromatography. Glycosides, phenols, steroids and hydrophilic 
compounds are often difficult to chromatograph using normal phase silica gel. During 
the processing thousands of pounds of bark and lesser amounts of needles many 
interesting compounds were isolated. The taxane glycosides are most notable among 
these, especially now that the efficient removal of the glycosyl moiety has become 
possible (Rao, 1997). The relative abundance of taxane glycosides amenable to 
conversion into taxol gives further support to the use of reverse phase columns. Large 
amounts of valuable precursors are lost with the normal Polysciences isolation process 
(Boettner ef a/. 1979). 



Flavonoids are chemicals generally found in plants that are ubiquitous and have 
been studied for hundreds of years. These compounds are generally yellow to red in 
color and are usually responsible for the colors seen in flowers and plants. Research 
into the possibility that flavonoids might possess useful biological activities has 
undergone a renaissance in recent years, after decades of neglect. Quercetin and its 
most common glycoside (rutin) are probably the most ubiquitous of the flavonoids, and 
have been used for many years to enhance immune response to pathogens. 
"Bioflavonoid complex" is mostly rutin and another flavonoid known as hesperidin, and 
can be purchased in most drug stores and herbal shops. 

These flavonoid compounds were easily purified on the reverse phase columns. 
The future usefulness of flavonoids as bioactive compounds remains to be seen, but the 
evidence is growing. Some of these compounds have been reported to inhibit the 
reverse transcriptase enzyme of HIV virus in vitro, but further work is needed concerning 
the mechanism. 

Insect molting hormones, commonly classed as "edcysones," are responsible for 
the maturation of larvae into adult form and have interested scientists for many decades. 
Originally, silk worms were extracted to obtain these compounds for research and in 
very low yields indeed. Efforts to find better sources for these compounds led to their 
discovery in some plants, including Taxus baccate (Hoffmeister 1966). Substantial 
amounts of (3-ecdysterone, ponasterone A and other ecdysones were easily isolated 
during the workup of fractions from the reverse phase columns. All samples tested in 
this project had substantial amounts of these hormones, and were easy to isolate. 

Usnic acid is a bright yellow compound which is known to grow in lichens. Due 
to its structure all signals in the proton NMR are singlets. It is commonly classed as a 
tricarbonyl and has peaks as high as 18.8, 13.3 and 11 ppm. An endophytic fungus of 
the pacific yew known as Taxomyces andreanae is capable of producing taxol in cell 


culture (Stierle et al. 1993). It is a mystery how these two organisms that are so different 
are able to make a complex diterpene like taxol. In spite of this taxol producing fungus, 
it is most likely that the usnic acid was actually produced by lichens growing on the bark, 
and is not produced by the tree. Usnic acid is mentioned here because it was difficult to 
characterize and not uncommon in samples processed in this lab. Usnic acid is also 
known to be quite toxic and should be handled carefully. 

Betuloside is a simple glycoside first isolated from the plant Betula pendata 
(Khan 1966). Animal studies using hepatotoxic agents indicate that betuloside has 
significant hepatoprotective activity. The mechanism by which this compound protects 
the liver is not known, but teas made from plants containing betuloside have been used 
in India for centuries for various problems. Betuloside is just one more example of the 
usefulness of preparative scale reverse phase chromatography. 


Analytical HPLC was performed using two different systems. For determinations 
of purity and quantitative information on composition, a setup with a Waters 600 E pump 
with gradient control, a Waters 996 photodiode array detector, and a Waters 717 
autosampler, coupled with an NEC-386 computer and printer was used. Waters 
Millennium 1.1 program was used with the photodiode array system. For routine use, a 
combination of a Waters 501 pump with a U6K injector, a 486 tunable absorbance 
detector and a Goerz Servogor 120 recorder was used. 

For analytical purposes, standard columns packed with C-8 bonded silica 
(Whatman Partisil®, 4.6 mm x 25 cm, 5f.L) were used with either of the solvents: 50% 
acetonitrile in water, or a 5:4:1 mixture of acetonitrile, water and methanol. 

For preparative scale purposes, stainless steel columns of two sizes were used: 
4" x 4' and 6" x 6', fabricated by Fluitron Inc. (Ivyland, PA) and rated to 200 psi. The 


columns were packed with C-18 or C-8 bonded silica gel (Spherisorb, 15-35 |li diameter, 
Phase Separations Inc., Nonwalk, CT) as a slurry in methanol . After thorough washing 
with methanol, the columns were equilibrated with 25% acetonitrile in water. 

Thin-layer chromatography was carried out using silica gel HF-60, 254+366 (EM 
Science/Fisher). Visualization was by a UV-lamp and by charring with 1 N H2SO4. 
Column chromatography was performed using silica gel (Fisher, 100-200 and 235-425 
mesh) or Florisil (Fisher F-101, 100 mesh) with a solvent sequence consisting of 
ligroin/CHCbCHCIs, 2-5% acetone and finally, 2-10% MeOH in CHCI3. 

Melting points were determined on a Fisher-Johns hot stage apparatus and are 
uncorrected. The following instrumentation was used for the spectra recorded here: 
UV, Perkin Elmer 13B; IR, Perkin Elmer PE-1420; and NMR, General Electric QE-300, 
Nicolet NY-300, Varian VXR-300, Varian Gemini-300 and Varian Unity-600 
spectrometers. Mass spectra (FAB) were obtained on a Finnegan Mat 95Q 
spectrometer using a cesium gun operated at 15 KeV of energy. 


Quercetin Rutoside (Rutin) 

dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one. m.p. : 186-189 °C (dec, turns 
brown at~127°C); Md'' +14.06 (ethanol, c 1.02); [ab'' -39.76 (pyridine, c 1.06). 

^H NMR (DMSO-ds, 300 MHz, 5); 12.6 (C-5 hydroxyl, br s, D2O exchangeable); 
7.57 (H-2', d, 2.2); 7.53 (H-6', dd, 8.6, 2.2); 6.86 (H-5', d, 8.3); 6.40 (H-8, br s); 6.21 (H-6, 
br s); 5.34 (H-1" glucosyl, br s); 4.42 (H-7" rhamnosyl, br s); 3.1-3.75 (CjH-OH glycosyl, 
6H, mult.); 1.03 (H-12" methyl, d, 6.0). 


^^C NMR (DMSO-ds, 300 MHz, 5): 177.5 (C-4, carbonyl); 164.1 (C-7, s); 161.4 
(C-5, s); 156.9 (C-2, s); 156.7 (C-9, s); 148.5 (C-4', s); 144.8 (C-3',s); 133.5 (C-3, s); 
121.9 (C-6', d); 121.4 (C-1',s); 116.4 {C-2',d); 115.4 (C-5',d); 104.2 (C-10, s) ;100.6 (C- 
1",d); 98.6 (C-6,d); 93.5 (C-8, d); 76.5; 76.0; 74.2; 71.9; 70.6; 70.55; 70.49; 70.1; 68.4; 
67.2 (C-6", t); 18.0 (C-12" methyl, q). 

IR, V max (KBr, cm"'): 3340 (OH, bonded); 2920 (CH stretch); 1655 (C=0); 1620 
(C=C); 1510 (aromatic); 1355 (C-O-C); 1290 (C-O-C); 1200 (C-O-C); 1055 (C-O-C); 970, 
880, 810 (subst. aromatic); 730, 695. 

Analysis calculated for C27H30O16: C, 53.12; H, 4.95. Found: C, 52.88; H, 5.06. 

2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one or 3,3', 4', 5,7- 
pentahydroxyflavone. Refluxed 300 mg of quercetin glycoside in 2 N H2SO4 for 3 hours, 
filtered, washed with water, yellow needles crystallized from aqueous ethanol, 150 mg 

'H NMR (DMSO-ds, 300 MHz, 5): 12.5 (C-5 hydroxyl, br s, exchangeable with 
D2O); 7.68 (H-2', d, J=2.2); 7.54 (H-6', dd, J= 2.0 and 8.4); 6.90 (H-5', d, J=8.4); 6.41 
(H-8, d, J=2.0); 6.19 (H-6, d, J=2.4). 

^'C NMR (DMSO-ds, 300 MHz, 5): 175.7 (C-4, s); 163.8 (C-7, s); 160.7 (C-5, s); 
156.9 (C-9, s); 147.6 (C-4', s); 146.9 (C-2, s); 145.0 (C-3', s); 122.0 (C-1', s); 120.0 (C- 
6', d); 115.6(0-5', d); 115.3 (C-2', d); 103.0 (C-10, s); 98.2 (C-6, d); 93.4 (C-8, d). 

Analysis calculated for C15H10O7+ 2 H2O: C, 53.26; H, 4.17. Found: C, 53.64; H, 

This biflavonoid is a member of the amentoflavone group, m.p. : 302-304° C. 


^H NMR (DMSO-ds, 300 MHz, 5): 13.05 (1H, s, 5'-0H); 12.90 (1H, s, 5-OH); 8.31 
(1H, s, 7-OH); 8.17 (1H, dd, J=9.0, 2.4, 6'-H); 8.08 (1H, d, J=2.4, 2'-H); 7.60 (2H, d, 
J=9.0, 2'" H, 6'" H); 7.37 (1H, d, J=8.7, H-5'); 6.98 (1H, s. H-3"); 6.93 (2H, d, J=9, H-3'", 
H-5'"); 6.89 (1H, s); 6.78 (1H, d, J=2.4, H-8); 6.42 (1H, s, H-6"); 6.36 (1H, s, 6-H); 3.83 
(3H, s); 3.80 (3H, s); 3.76 (3H, s). 

"C NMR (DMSO-dg, 300 MHz, 8): 181.9; 181.8; 165.1; 163.5; 162.9; 162.1; 
161.0; 160.5; 160.4; 157.2; 154.2; 130.8; 128.2; 127.7; 122.7; 122.3; 121.5; 114.4; 
111.6; 104.1; 103.7; 103.55; 103.48; 103.1; 98.6; 98.0; 92.6; 55.9; 55.8; 55.4. 

IR, y max (KBr, cm ') : 1660, 1650, 1620, 1610, 1570, 1510, 1430, 1370, 1240, 
1180, 1160, 1050, 1030, 960, 910, 880, 830, 760. 

Analysis calculated for C33H24O10: C, 68.27; H, 4.17. Found: C, 67.94; H, 4.26. 

Identity of glycoside confirmed using autiientic sample with mixed melting point, 
TLC, and IR spectrum, as well as ^H- and "C NMR of the tetra-acetate, the aglycone, 
and the aglycone acetate. M.p. : 288-290 ° C (lit. varies from 280-300 ° C, 298 ° C 
Sucrow 1966). EI-MS: 414(2%) [M+ - glucosyl], 396(34%) [MH+ - glucosyl - H2O]. Cl- 
MS (methane): 413(9%) [MH+ - glucosyl - H2], 397(100%) [MH+ - glucosyl - H2O]. 
[a]D^' -40.1 (pyridine, c 1.1); (lit. -41.0, c 1.33, pyridine. Swift 1952). 

IR y max (KBr, cm"') : 3400 (-OH); 3090 (C=CH2); 1650, 890. 

Analysis calculated for CssHsoOb: C, 72.87; H, 10.48. Found: C, 72.48; H, 10.61. 
p-Sitosterol-B-D-Glucoside Tetra-acetate 

Acetylation of p-sitosterol-p-D-glucoside: Dissolved 500 mg in 5 ml acetic 
anhydride with 0.1 ml pyridine, then placed in hot water bath for 1 hour with stirring. 
After TLC indicated the reaction was complete the mixture was stirred with water for 15 
minutes to decompose the anhydride, then extracted with chloroform 3 x at pH 4. Dried 


over Na2S04, concentrated, then crystallized from ethyl acetate in ligroin, yield 440 mg 

; (68%); first crop. 

■ M.p.: 165-167 ° C (lit. 171 ° C, Sucrow 1966). [a^^^ -35.0 (pyridine, c 1.3); (lit. 

pyridine, c 1.33, -33.7, Swift 1952). 

'H NMR (CDCI3, 300 MHz, 5): 5.37 (H-6, br d, J= 4.8); 5.21 (H-3', t, J=9.3); 5.08 
(H-4', t, J=9.2); 4.95 (H-2', dd, J=9.3 and 8.1); 4.60 (H-r, d, J=8.1); 4.26 (H-6'b, dd, 
J=4.8 and 12); 4.11 (H-6'a, dd, J=2.4, 12); 3.70 (H-5', cm); 3.5 (H-3, cm); 2.07, 2.05, 
2.02, 2.00 (4 X Ac CH3, s); 1 .0 (H3C-1 9, s); 0.84 s; 0.82 s; 0.68 (H3C-1 8, s). 

''C NMR (CDCI3, 75 MHz, 5): 170.6, 170.3, 169.3, 169.2 (4 X AcC=0, s); 140.3 
(C-5, s); 122.1 (C-6, d); 99.6 (C-l', d); 80.0 (C-3, s); 72.9 (C-3', d); 71.7 (C-5, d); 71.5 
(C-2', d); 68.5 (C-4', d); 62.1 (C-6', t); 56.7 (C-14, d); 56.0 (C-17, d); 50.1 (C-9, s); 45.8 
(C-24, s); 42.3 (C-13, s); 39.7 (C-12, t); 38.9 (C-4, d); 37.2 (C-1, d); 36.7 (C-10, s); 36.1 
(C-20, d); 33.9 (C-22, t); 31.9 (C-8, d); 31.8 (C-7, t); 29.4 (C-2, t); 29.1 (C-25, d); 28.2 (C- 
16, t); 26.1 (C-23, t); 24.3 (C-1 5, d); 23.0 (C-28, t); 21.0(C-11, d); 20.6, 20.5, 20.44, 
20.42 (4 X AcMe); 19.7 (C-27, q); 19.3 (C-1 9, q); 19.0 (C-26, q); 18.7 (C-21, q); 11.8 (C- 
18, q); 11.8(C-29, q). 

IR, v/max(KBr, cm"^) : 1755, 1220, 1045, 910. 

M.p.: 138-140 ° C (lit. 137-138 °C, Swift 1952); [ab^' -37.2 (chloroform, c 1.1); 
(lit. -38.2, chloroform, c 5.1, Swift 1952). 

'H NMR (CDCI3, 300 MHz, 5): 5.36 (H-6, d); 3.50 (H-3, m); 2.30 m, 2.0 m, 1.0 s, 

0.82 d, 0.7 s. 

^'C NMR (CDCI3, 75 MHz, 5): 140.7 (C-5, s); 121.6 (C-6, d); 71.8 (C-3, s); 56.7 
(C-14, d); 56.0 (C-17, d); 50.1 (C-9, d); 45.8 (C-24, d); 42.3* (C-13, s); 43.2 (C-4, t); 39.7 
(C-12, t); 37.2 (C-1, t); 36.4 (C-10, s); 36.1 (C-20, d); 33.9 (C-22, t); 31.9 (C-8, d); 31.6 


(C-2, t); 29.2 (C-25, q); 28.2 (C-16, t); 26.1 (C-23, t); 24.3 (C-15, q); 23.0 (C-28, t); 21.0 
(C-11, t); 19.8 (C-27, q); 19.3 (C-19, q); 19.0 (C-26, q); 18.7 (C-21, q); 11.8 (C-18, q); 
11.9(C-29, q). 

Analysis calculated for C29H50O: C, 83.99; H, 12.15. Found: C, 83.16; H, 12.42. 


Ecdvsterone & 2B, 3B, 22a-Triacetate 

(22R)-2p,3p,14a,20p,22a,25-hexahydroxycholest-7-en-6-one, m.p.: 237-240 ° C 
(Lit. 240 ° C, Takemoto et al. 1967). 

'H NMR (CDCI3, Triacetate, 5): 5.85 (1 H, s, H-7); 5.31 (1 H, br s, H-3a); 5.04 (1 H, 
dt=9, 3 Hz, H-2a); 4.79 (1H, d=9 Hz, H-22P); 3.10 (1H, br t=7.8 Hz, H-9a); 2.10 (6H, s, 
2X OAc); 1.99 (3H, s, OAc); 1.26 (3H, s, Me-21); 1.23 (3H, s, Me-26*); 1.21 (3H, s, C- 
27); 1.04 (3H, s, Me-19); 0.85 (3H, s, IVIe-18). 

^'C NIVIR (CDCI3, Triacetate, 5): 202.1 (C-6); 172.2 (OAc); 170.3 (OAc); 170.0 
(OAc); 164.8 (C-8); 121.6 (C-7); 84.4 (C-14); 79.8 (C-22); 77.0 (C-20); 70.4 (C-25); 68.7 
(C-3); 67.2 (C-2); 51.0 (C-5); 49.6 (C-17); 47.6 (C-13); 40.4 (C-24); 38.4 (C-l*); 38.3 (C- 
10*); 34.1 (C-4); 33.7 (C-9); 31.5 (C-15); 31.2 (C-12); 30.2 (C-27); 29.2 (C-16); 28.5 (C- 
26); 24.7 (C-23); 23.8 (C-19); 20.6 (C-21t); 20.5 (C-11t); 17.5 (C-18). 

IR, vmax (KBr, cm^^): : 3400, 2960, 2870, 1643, 1450, 1370, 1050, 880. 

UV max (ethanol): 243 nm (s 10,400) 

Analysis calculated forC27H4407: C, 67.47; H, 9.23. Found: C, 67.08; H, 9.30. 
Ponasterone A and 2P. 3P, 22a Triacetate 

(22R)-2p, 3p, 14a, 20p, 22a-pentahydroxycholest-7-en-6-one. M.p. : 256-262 °C 
(Lit. 259-260 ° C, Nakanishi et al. 1966) 


^H NMR (CDCI3, Triacetate, §): 5.87 (1H, s, H-7); 5.31 (1H, br s, H-3a); 5.04 (1H, 
dt=9, 3 Hz, H-2a); 4.84 (1H, d=9 Hz, H-22p); 3.12 (1H, br t=7.8 Hz, H-9a); 2.11 (6H, s, 
2X OAc); 2.01 (3H, s, OAc); 1.25 (3H, s, Me-21); 1.03 (3H, s, Me-19); 0.89 (3H, d=2.7 
Hz, Me-26*); 0.87 (3H, d=2.7 Hz, Me-27*); 0.85 (3H, s, Me-18). 

''C NMR (CDCI3, Triacetate, 5): 201.9 (C-6); 172.3 (OAc); 170.4 (OAc); 170.1 
(OAc); 164.7 (C-8); 121.5 (C-7); 84.4 (C-14); 79.5 (C-22); 76.9 (C-20); 68.7 (C-3); 67.1 
(C-2); 50.9 (C-5); 49.6 (C-17); 47.5 (C-13); 38.3 (C-1); 35.7 (C-10); 34.1 (C-4); 33.6 (C- 
9); 31.5 (C-1 5); 31.2 (C-1 2); 29.2 (C-1 6); 27.9 (C-23); 27.7 (C-25); 23.8 (C-21); 22.9 (C- 
19); 22.1, 21.1 (C-27*); 21.0 (C-26*); 20.6 (C-24t); 20.5 (C-11t); 17.4 (C-18). 

IR, \/max (KBr, cm'^) : 3400, 2960, 2870, 1643, 1450, 1380, 1050, 870. 

Analysis calculated forC27H4406: C, 69.79; H, 9.54. Found: C, 69.41; H, 9.72. 

Phenolic Compounds 

Usnic Acid 

m.p. : 208-213° C, yellow orthorhombic prisms from ligroin/ethyl acetate (lit. 204, 
acetone, Schopf & Ross, 1938). Hd'' -510, CHCI3, c 0.62 (lit. -509, CHCI3, c 0.679, 
Schopf & Ross, 1938). 

^H NMR (CDCI3, 300 MHz, 5): 1.76 (3H, s, C-4' angular methyl); 2.10 (3H, s, C-6 
aromatic methyl); 2.67 (6H, s, C-3 & C-8 acetyl methyls); 5.97 (1H, s, C-1); 11.02 (1H, s, 
C-5 phenol); 13.30 (1H, s, C-7 phenol); 18.83 (1H, s, C-4 enol). 

^^C NMR (CDCI3, 75 MHz, S): 7.5 (q, C-6 methyl); 27.9 (q, C-4' angular methyl); 
31.3 (q, C-8 acetyl methyl); 32.1 (q, C-3 acetyl methyl); 59.0 (s, C-4'); 98.3 (d, C-1); 
101.5 (s, C-3); 103.9 (s, C-8); 105.2 (s, C-6); 109.2 (s, C-5'); 155.1 (s, C-8'); 157.4 (s, C- 
7); 163.8 (s, C-5); 179.0 (s, C-1'); 191.6 (s, C-8 acetyl CO); 198.0 (s, C-2 CO); 200.3 (s, 
C-3 acetyl CO); 201.7 (s, C-4 CO). 


IR, v max (KBr, cm"^): 1690 (dienone carbonyl at C-2); 1630 (aromatic C-acetyl); 
1610 (enol ether and aromatic double bonds); 1540 (conjugate carbonyl at C-3). 

Analysis calculated for CisHieOy: C, 62.77; H, 4.69. Found: C, 62.47; H, 4.75. 
Betuloside (4-(4'-Hvdroxvphenvl)-2R-butanol Glucoside)) & Aqlycone 

M.p. : 191-193 °(Lit. 187-190 ° Khan ef a/. 1976). 

^H NMR (CDCI3, 5): 1.17 (3H, d, Me, J=5.7 Hz); 1.8 (2H, cm, H-3); 2.58 (2H, t, H- 
4, J=7.5 Hz); 3.6-4.5 (glucosyl); 6.8 & 7.0 (4H, d, A2B2 Aromatic, J=8.2 Hz); 8.84 (1H, s, 

"C NMR (CDCI3, aglycone, 5): 153.9 (C-4'); 133.7 (C-l'); 129.4 (C-2', 6'); 115.3 
(C-3', 5'); 67.8 (C-2); 40.7 (C-3); 31.2 (C-4); 23.4 (C-1 Me). 

IR, V max (KBr, cm"'): 3370, 2930, 2860, 1610, 1590, 1510, 1435, 1445, 1370, 

Analysis calculated for C10H14O2: C, 72.26; H, 8.49. Found: C, 72.12; H, 8.56. 


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Richard Michael Davies was born in Cocoa, Florida on March 21, 1959 to Dan 
and Ruth Lewis Davies. He attended Rockledge High School where he participated in 
the cross country and track teams, the school's concert and marching bands, and other 
extracurricular activities and societies. He attended Brevard Community College for one 
year before enrolling at the University of Florida to complete bachelor's degrees in 
chemistry and then pharmacy. While studying pharmacy he also worked on projects 
with Professor Rao in the laboratory. Interest in the chemistry and research of natural 
products and the development of new anticancer therapies brought him back for 
graduate studies. 


I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quality, as a dissertation for the degree of Doctor of Philosophy. 

Koppaka V. Rao, Chair (deceased) 
Professor of Medicinal Chemistry 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quality, as a dissertation for the degree of Doctor of Philosophy. 


Perrin, Cochair 
ssor of Medicinal Chemistry 

I certify that 1 have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quality, as a dissertation for the degree of Doctor of Philosophy. 

Margaretp. James 

Professor of Medicinal Chemistry 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quality, as a dissertation for the degree of Doctor of Phijosophy. 

Kenneth B. Sloan 

Professor of Medicinal Chemistry 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quality, as a dissertation for the degree of Doctor of Philosophy. 

U-^' "^^^-^^-^-^ 

Jonathan Eric Enholm 
Associate Professor of Chemistry 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate^scope - 
quality, as a dissertation for the degree of Doctpr'orCTilosojahy. 

Jicinai Chemistry 

This dissertation was submitted to tiie Graduate Faculty of tine College of 
Pharmacy and to the Graduate School and was acc^tpd as p^i^i^ fulfillment qt±£ie 
requirements for the degree of Doctor of Philosopj; 

December 1998 

Dean, Colrege oil Pharmacy 

Dean, Graduate School